Methods and compositions for wound healing

ABSTRACT

Methods and compositions comprising combinations of one or more anti-connexin agents and one or more other agents useful for the promotion and/or improvement of wound healing and/or tissue repair.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/590,113 filed on Aug. 20, 2012 and will issue as U.S. Pat. No.9,457,044 on Oct. 4, 2016, which is a continuation of and claimspriority to U.S. patent application Ser. No. 11/985,717 filed on Nov.15, 2007 and issued as U.S. Pat. No. 8,247,384 on Aug. 21, 2012, theentire contents of which is hereby incorporated by reference in itsentirety, which claims the benefit of U.S. Provisional Application Ser.No. 60/859,437, filed on Nov. 15, 2006, the entire contents of which ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The field relates to wound-healing and tissue repair, and to connexins,connexin hemichannels and gap junctions, including compositions with oneor more anti-connexin agents and one or more therapeutic agents, agentsuseful for wound healing, and/or gap junction modifying agents, articlesand kits and delivery devices containing such compositions, andformulations comprising such compositions, as well as methods oftreating wounds and diseases, disorders or conditions characterized inwhole or in part by acute, delayed or incomplete wound healing or whichwould benefit from improved tissue repair or healing.

BACKGROUND AND INTRODUCTION TO THE INVENTION

The following includes information that may be useful in understandingthe present inventions. It is not an admission that any of theinformation provided herein is prior art, or relevant, to the presentlydescribed or claimed inventions, or that any publication or documentthat is specifically or implicitly referenced is prior art.

In humans and other mammals wound injury triggers an organized complexcascade of cellular and biochemical events that will in most casesresult in a healed wound. An ideally healed wound is one that restoresnormal anatomical structure, function, and appearance on cellular,tissue, organ, and organism levels. Wound healing, whether initiated bytrauma, microbes or foreign materials, proceeds via a complex processencompassing a number of overlapping phases, including inflammation,epithelialization, angiogenesis and matrix deposition. Normally, theseprocesses lead to a mature wound and a certain degree of scar formation.Although inflammation and repair mostly occur along a prescribed course,the sensitivity of the process is dependent on the balance of a varietyof wound healing molecules, including for example, a network ofregulatory cytokines and growth factors.

Gap junctions are cell membrane structures that facilitate directcell-cell communication. A gap junction channel is formed of twoconnexons (hemichannels), each composed of six connexin subunits. Eachhexameric connexon docks with a connexon in the opposing membrane toform a single gap junction. Gap junction channels are reported to befound throughout the body. Tissue such as the corneal epithelium, forexample, has six to eight cell layers, yet expresses different gapjunction channels in different layers with connexin 43 in the basallayer and connexin 26 from the basal to middle wing cell layers. Ingeneral, connexins are a family of proteins, commonly named according totheir molecular weight or classified on a phylogenetic basis into alpha,beta, and gamma subclasses. At least 20 human and 19 murine isoformshave been identified. Different tissues and cell types are reported tohave characteristic patterns of connexin protein expression and tissueshave been shown to alter connexin protein expression pattern followinginjury or transplantation (Qui, C. et al., (2003) Current Biology,13:1967-1703; Brander et al., (2004), J. Invest Dermatol. 122:1310-20).

Antisense technology has been proposed for the modulation of theexpression for genes implicated in viral, fungal and metabolic diseases.See, for example, U.S. Pat. No. 5,166,195, (oligonucleotide inhibitorsof HIV) and U.S. Pat. No. 5,004,810 (oligomers for hybridizing to herpessimplex virus Vmw65 mRNA and inhibiting replication). See also U.S. Pat.No. 7,098,190 issued to Becker and Green (“Formulations comprisingantisense nucleotides to connexins”). Peptide inhibitors of gapjunctions and hemichannels have also been reported. See for exampleBerthoud, V. M. et al., Am J. Physiol. Lung Cell Mol. Physiol. 279:L619-L622 (2000); Evans, W. H. and Boitano, S. Biochem. Soc. Trans. 29:606-612, and De Vriese A. S., et al. Kidney Int. 61: 177-185 (2001). Seealso Becker and Green PCT/US06/04131 (“Anti-connexin compounds and usesthereof”).

Various cytokines and growth factors have been investigated to determinetheir potential as therapeutic interventions in wound healing. Saveplatelet-derived growth factor, however, the active ingredient inRegranex®, none have been approved for sale in the United States. And,despite advances in the understanding of the principles underlying thewound healing process, there remains a significant unmet need forsuitable therapeutic options for wound care and improving and/orpromoting wound healing, including delayed or compromised wound healingsuch as chronic wounds, as well treatment of swelling, inflammation, andscarring associated with wounds, including acute and subacute wounds.

BRIEF SUMMARY

The inventions described and claimed herein have many attributes andembodiments including, but not limited to, those set forth or describedor referenced in this Brief Summary. It is not intended to beall-inclusive and the inventions described and claimed herein are notlimited to or by the features or embodiments identified in this BriefSummary, which is included for purposes of illustration only and notrestriction.

The invention generally relates to the use of one or more anti-connexinagents (for example, connexin inhibitors such as alpha-1 connexinoligodeoxynucleotides and alpha-1 anti-connexin peptides orpeptidomimetics) in combination with one or more therapeutic agents,agents useful for wound healing, and/or gap junction modifying agentsfor the treatment of wounds, including acute, subacute, delayed healingand chronic wounds.

The present invention provides for an increase in the rate, extentand/or quality of wound healing through the use of one or moreanti-connexin agents and one or more therapeutic agents, agents usefulfor wound healing, and/or gap junction modifying agents. In a preferredembodiment, the combined use of one or more anti-connexin agents and oneor more therapeutic agents, agents useful for wound healing, and/or gapjunction modifying agents has an additive, synergistic or super-additiveeffect in the promotion of wound healing. In another preferredembodiment, the combined use of one or more anti-connexin agents and oneor more therapeutic agents, agents useful for wound healing, and/or gapjunction modifying agents allows the use of reduced doses of such agentscompared to the dose or doses that may be effective when the agent isadministered alone. In another preferred embodiment, the combined use ofone or more anti-connexin agents and one or more therapeutic agents,agents useful for wound healing, and/or gap junction modifying agentsallows a reduced frequency of administration compared to the frequencyof administration when the agent is used alone.

Compositions and methods of the invention that employ anti-connexinagents in combination with other therapeutic agents, agents useful forwound healing, and/or gap junction modifying agents are disclosed andclaimed.

The invention includes pharmaceutical compositions comprising (a) atherapeutically effective amount of an anti-connexin agent, and (b) atherapeutically effective amount of another therapeutic agent useful inthe treatment of wounds or the promotion of wound-healing. The inventionincludes pharmaceutical compositions comprising (a) a therapeuticallyeffective amount of an anti-connexin agent, and (b) a therapeuticallyeffective amount of a gap junction modifying agent. Preferably, thepharmaceutical compositions further comprise a pharmaceuticallyacceptable carrier, diluent or excipient.

Pharmaceutical compositions are provided for combined, simultaneous,separate sequential or sustained administration. In one embodiment, acomposition comprising one or more anti-connexin agents is administeredat or about the same time as one ore more therapeutic agents, agentsuseful for wound healing, and/or gap junction modifying agents

Pharmaceutical compositions are also provided in the form of a combinedpreparation, for example, as an admixture of one or more anti-connexinagents and one or more other agents useful for wound healing, e.g.,growth factors that are effective in promoting or improving woundhealing, such as platelet derived growth factor, epidermal growthfactor, fibroblast growth factor (e.g., FGF2), vascular endothelialgrowth factor, and transforming growth factor β3, and/or cytokines thatare effective in promoting or improving wound healing, such as IL-7 andIL-10, and/or other agents that are effective in promoting or improvingwound healing, such as IGF (e.g., IGF-1) and IGFBP (e.g., IGFBP-2).

The term “a combined preparation” includes a “kit of parts” in the sensethat the combination partners as defined above can be dosedindependently or by use of different fixed combinations withdistinguished amounts of the combination partners (a) and (b), i.e.simultaneously, separately or sequentially. The parts of the kit canthen, for example, be administered simultaneously or chronologicallystaggered, that is at different time points and with equal or differenttime intervals for any part of the kit of parts.

In a preferred embodiment, the administration of a combined preparationwill have fewer administration time points and/or increased timeintervals between administrations as a result of such combined use.

In one aspect, the invention includes pharmaceutical compositions,including topical delivery forms and formulations, comprising apharmaceutically acceptable carrier and therapeutically effectiveamounts of an anti-connexin agent and one or more agents describedherein. Examples of anti-connexin agents include anti-connexinoligodeoxynucleotides (“ODN”), including antisense (including modifiedand unmodified backbone antisense), RNAi, and siRNA, as well asanti-connexin peptides and peptidomimetics. Suitable anti-connexinagents include for example, antisense ODNs, peptides and peptidomimeticsagainst connexins 43, 26, 37, 30, and 31.1 and 32. In certainembodiments, suitable compositions include multiple anti-connexin agentsin combination, including for example, connexin 43, 26, 30, and 31.1,together with one or more other agents useful in wound healing and/ortissue repair. Preferred anti-connexin agents are directed againstconnexin 43. Preferred agents for use in wound healing in combinationwith one or more anti-connexin agents include certain growth factorsincluding, for example, platelet derived growth factor, epidermal growthfactor, fibroblast growth factor alpha, fibroblast growth factor beta,vascular endothelial growth factor, and transforming growth factor β3,as well as insulin-like growth factor. Other preferred agents for use inwound healing in combination with one or more anti-connexin agentsinclude certain cytokines including, for example IL-7 and IL-10. Otherpreferred agents for use in wound healing in combination with one ormore anti-connexin agents include thymosin beta-4, secretory leukocyteprotease inhibitor, beta adrenergic antagonists (e.g., timoptic),interleukin-1 receptor antagonists (e.g., anakinra), free radicalscavengers (e.g., N-acetylcysteine), and gene therapy vectors comprisinga coding sequence for a protein useful in the promotion or improvementof wound healing (e.g., an adenovirus vector including a sequence codingfor platelet derived growth factor-B). Preferred therapeutic agentsadministered in combination with one or more anti-connexin agentsinclude, for example anti-inflammatory agents, antimicrobial agents(e.g., trimethoprim), local and topical anesthetics, and topical opioids(e.g., morphine, hydromorphone and fentanyl).

In another aspect, the invention includes methods for administering atherapeutically effective amount of one or more pharmaceuticallyacceptable anti-connexin agent and one or more therapeutic agents,agents useful for wound healing and/or gap junction modifying agentsformulated in a delayed release preparation, a slow release preparation,an extended release preparation, a controlled release preparation,and/or in a repeat action preparation to a subject with a wound,including wounds characterized in whole or in part by delayed orincomplete wound healing.

In certain other aspects, the invention also relates to methods of usingsuch compositions to treat subjects suffering from or at risk forvarious diseases, disorders, and conditions associated with a wound,including acute and subacute wounds, and delayed healing or chronicwounds.

In yet another aspect, the invention includes methods for treating asubject having or suspected of having or predisposed to, or at risk for,any diseases, disorders and/or conditions characterized in whole or inpart by a wound or a tissue in need of repair. Such compositionsinclude, for example, topical delivery forms and formulations.

Preferred methods include the sequential or simultaneous administrationof one or more anti-connexin agents and one or more agents useful forwound healing, either or both of which are provided in amounts or dosesthat are less that those used when the agent or agents are administeredalone, i.e., when they are not administered in combination, eitherphysically or in the course of treatment of a wound. Such lesser amountsof agents administered are typically from about one-twentieth to aboutone-tenth the amount or amounts of the agent when administered alone,and may be about one-eighth the amount, about one-sixth the amount,about one-fifth the amount, about one-fourth the amount, about one-thirdthe amount, and about one-half the amount when administered alone.Preferably, the administration is sequential. Preferably, the agents areadministered sequentially within at least about one-half hour of eachother. The agents may also be administered with about one hour of eachother, with about one day to about one week of each other, or asotherwise deemed appropriate. Preferably, the anti-connexin agent isadministered first. Preferably, where one or more anti-connexin agentsare used, an anti-connexin peptide or anti-connexin peptidomimetic,e.g., an anti-connexin agent that can block or reduce hemichannelopening, is administered prior to the administration of an anti-connexinagent that blocks or reduce connexin expression or the formation ofhemichannels or gap junctions, e.g., by downregulation of connexinprotein expression. Preferably, the anti-connexin agent or agents is/areanti-connexin 43 agent(s).

In a further aspect, the invention includes transdermal patches,dressings, pads, wraps, matrices and bandages capable of being adheredor otherwise associated with the skin of a subject, said articles beingcapable of delivering a therapeutically effective amount of one or morepharmaceutically acceptable anti-connexin agents and one or moreadditional pharmaceutically acceptable therapeutics agents, agentsuseful for wound healing, and/or gap junction modifying agents to asubject.

In another aspect, the invention includes an article of manufacturecomprising a vessel containing a therapeutically effective amount of oneor more pharmaceutically acceptable anti-connexin agents and one or morepharmaceutically acceptable therapeutic agents for promotion of woundhealing and instructions for use, including use for the treatment of asubject.

In another aspect, the invention includes an article of manufacturecomprising a vessel containing a therapeutically effective amount of oneor more pharmaceutically acceptable anti-connexin agents and one or morepharmaceutically acceptable agents useful for wound healing forpromotion of wound healing and instructions for use, including use forthe treatment of a subject.

In another aspect, the invention includes an article of manufacturecomprising a vessel containing a therapeutically effective amount of oneor more pharmaceutically acceptable anti-connexin agents and one or morepharmaceutically acceptable gap junction modifying agents andinstructions for use, including use for the treatment of a subject.

The invention includes an article of manufacture comprising packagingmaterial containing one or more dosage forms containing one or morepharmaceutically acceptable anti-connexin agents and one or morepharmaceutically acceptable therapeutic agents, agents useful for woundhealing, and/or gap junction modifying agents, wherein the packagingmaterial has a label that indicates that the dosage form can be used fora subject having or suspected of having or predisposed to any of thediseases, disorders and/or conditions described or referenced herein,including diseases, disorders and/or conditions characterized in wholeor in part by acute, impaired, delayed or chronic wound healing. Suchdosage forms include, for example, topical delivery forms andformulations.

The invention includes a formulation comprising a pharmaceuticallyacceptable anti-connexin agent and a pharmaceutically acceptable agentuseful for wound healing in amounts effective to promote healing ortissue repair in a subject. The invention includes a formulationcomprising a pharmaceutically acceptable anti-connexin agent and apharmaceutically acceptable therapeutic agent in amounts effective topromote wound healing in a subject. The invention includes a formulationcomprising a pharmaceutically acceptable anti-connexin agent and apharmaceutically acceptable gap junction modifying agent in amountseffective to promote wound healing in a subject. Such formulationsinclude, for example, topical delivery forms and formulations. Preferredformulations include one or more anti-connexin agents and one or moreagents useful for wound healing, either or both of which are provided inamounts or doses that are less that those used when the agent or agentsare administered alone, i.e., when they are not administered incombination, either physically or in the course of treatment of a wound.Such lesser amounts of agents administered or provided in combinationare typically from about one-twentieth to about one-tenth the amount oramounts when administered alone, and may be about one-eighth the amount,about one-sixth the amount, about one-fifth the amount, about one-fourththe amount, about one-third the amount, and about one-half the amountwhen administered alone.

The invention includes methods for the use of therapeutically effectiveamounts of compositions comprising one or more pharmaceuticallyacceptable anti-connexin agents and one or more pharmaceuticallyacceptable therapeutic agents, agents useful for wound healing and/orgap junction modifying agents in the manufacture of a medicament. Suchmedicaments include, for example, topical delivery forms andformulations. Such medicaments include those for the treatment of asubject as disclosed herein. Such medicaments preferably include thereduced amounts of the one or more anti-connexin agents and the one ormore pharmaceutically acceptable therapeutic agents, agents useful forwound healing and/or gap junction modifying agents, as noted herein.

The invention includes methods for the use of a therapeuticallyeffective amount of one or more anti-connexin agents and one or morepharmaceutically acceptable therapeutic agents, agents useful for woundhealing and/or gap junction modifying agents in the manufacture of adosage form. Such dosage forms include, for example, topical deliveryforms and formulations. Such dosage forms include those for thetreatment of a subject as disclosed herein. Such dosage forms preferablyinclude the reduced amounts of the one or more anti-connexin agents andthe one or more pharmaceutically acceptable therapeutic agents, agentsuseful for wound healing and/or gap junction modifying agents, as notedherein.

In another aspect, the invention provides method of treatment comprisingadministering to a subject a pharmaceutical composition of the inventionfor use in the treatment of a wound, including for example, acute,subacute, delayed and chronic wounds.

In another aspect, the invention provides for the use of ananti-connexin agent (for example, anti-alpha-1 ODN, peptide orpeptidomimetic) and a therapeutic agent, agent useful for wound healing,and/or gap junction modifying agent in the manufacture of apharmaceutical product for the promotion of wound healing in a patientin need thereof.

In yet another aspect, the invention provides a method of promoting orenhancing wound healing or treatment, or the prevention or ameliorationof fibrosis or other fibrotic conditions, the method comprisingadministering one or more anti-connexin agent and one or moretherapeutic agents, agents useful for wound healing and/or gap junctionmodifying agents to a patient in need thereof

In certain other aspect, the invention provides: (i) a packagecomprising an anti-connexin agent together with instructions for use incombination with a therapeutic agents, agents useful for wound healingand/or gap junction modifying agents for the promotion (e.g. decrease inhealing time, better wound outcome) of wound healing, (ii) a packagecomprising one or more therapeutic agents, agents useful for woundhealing and/or gap junction modifying agents together with instructionsfor use in combination with one or more anti-connexin agents for thepromotion of wound healing; and (iii) a package comprising one or moreanti-connexin agents and one or more therapeutic agents, agents usefulfor wound healing and/or gap junction modifying agents, together withinstructions for use in the promotion of wound healing or reduction ofwound-associated fibrosis.

In a one embodiment the pharmaceutical product of the invention isprovided in combination with a wound dressing or wound healing promotingmatrix. Suitably the wound dressing or matrix is provided including theform of a solid substrate with an anti-connexin agent and one or moretherapeutic agents, agents useful for wound healing and/or gap junctionmodifying agents dispersed on or in the solid substrate.

The anti-connexin agent and therapeutic agents, agents useful for woundhealing and/or gap junction modifying agents may be administered in thesame composition or by separate compositions. Preferably, the agents areadministered in the reduced amounts of the one or more anti-connexinagents and the one or more pharmaceutically acceptable therapeuticagents, agents useful for wound healing and/or gap junction modifyingagents, as noted herein.

The anti-connexin agent and therapeutic agents, agents useful for woundhealing and/or gap junction modifying agents may be administered to thepatient simultaneously, sequentially or separately. If administeredseparately, preferably the anti-connexin agent and the therapeuticagent, agent useful for wound healing and/or gap junction modifyingagent are administered sequentially. Preferably, the agents areadministered sequentially within at least about one-half hour of eachother. The agents may also be administered with about one hour of eachother, with about one day to about one week of each other, or asotherwise deemed appropriate. Preferably, the anti-connexin agent isadministered first. Preferably, where one or more anti-connexin agentsare used, an anti-connexin peptide or anti-connexin peptidomimetic,e.g., an anti-connexin agent that can block or reduce hemichannelopening, is administered prior to the administration of an anti-connexinagent that blocks or reduce connexin expression or the formation ofhemichannels or gap junctions, e.g., by downregulation of connexinprotein expression. Preferably, the anti-connexin agent or agents is/areanti-connexin 43 agent(s).

These and other aspects of the present inventions, which are not limitedto or by the information in this Brief Summary, are provided below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A to FIG. 1I depict expression of Cx43 at wound sites. FIG. 1Adepicts Real-Time PCR analysis of the gene expression of Cx43 at woundsites. FIG. 1A depicts relative expression levels of Cx43 to GAPDH ondays 1 and 7 in wounds treated with control sODN (n=4; open bars) andCx43 asODN (n=4; filled bars); data are expressed as the mean±s.e.m.*P<0.05. FIGS. 1B to 1F depict Cx43 staining (green) with bis-benzimidenuclear staining (blue) of wounds treated with Cx43 asODN (FIG. 1B: 1day, FIG. 1D: 2 days and FIG. 1F: 7 days) or controls (FIG. 1C: 1 day,FIG. 1E: 2 days and FIG. 1G: 7 days). FIGS. 1H and 1I depictillustration of sites imaged in the wound edge. (FIG. 1H) images B-E.(FIG. 1I) images F and G.

FIG. 2A to FIG. 2H depict cell proliferation after wounding. FIGS. 2A to2D depict analysis of cell proliferation at wound sites byimmunohistochemical staining with the anti-BrdU monoclonal antibody incontrol ODN (FIG. 2A: day 2 and FIG. 2C: day 7) and Cx43 asODN (FIG. 2B:day 2 and FIG. 2D: day 7). Arrowhead and arrow indicate the wound marginand leading edge, respectively. FIGS. 2E to 2H depict BrdU-stainedcells; (i) and the number of BrdU-positive cells per field in the woundmargin in the epidermis (FIG. 2E: n=5), and the nascent epidermis (FIG.2F: n=5); (ii) the number of BrdU-positive cells in the dermal woundedge (FIG. 2G: n=5) and in the forming granulation tissue (FIG. 2H:n=5). Counts are expressed as the mean±s.e.m. *P<0.05. Scale barsrepresent 200 μm.

FIG. 3A to FIG. 3C depict neutrophil recruitment into a wound site. FIG.3A and FIG. 3B depict neutrophil recruitment into skin wounds treatedwith control sODN (FIG. 3A) and Cx43 as ODN-treated (FIG. 3B), analyzedusing an anti-MPO antibody on day 1. FIG. 3C depicts numbers of MPOpositive cells at the wound site after treatment with control sODN (openbars: n=4 on day 1; n=5 on day 2) and Cx43 asODN (filled bars; n=3 onday 1; n=4 on day 2). Data are expressed as the mean±s.e.m. *P<0.05.Scale bars represent 50

FIG. 4A to FIG. 4C depict macrophage recruitment into a wound site. FIG.4A and FIG. 4B depict macrophage recruitment into skin wounds treatedwith control sODN (FIG. 4A) and Cx43 asODN (FIG. 4B), analyzed using ananti-F4/80 antibody on day 7. FIG. 4C depicts macrophage recruitmentinto skin wounds on days 2 and 7 after treatment with control sODN (openbars: n=4 on day 2; n=7 on day 7) and Cx43 asODN (filled bars: n=4 onday 2; n=6 on day 7). Data are expressed as the mean±s.e.m. *P<0.01.Scale bars represent 50

FIG. 5A to FIG. 5B depict expression of Cc12 and TNF-α at wound sites.FIG. 5A and FIG. 5B depict Real-Time PCR analysis of the gene expressionof Ccl2 and TNF-α at wound sites. Relative expression levels of Ccl2(FIG. 5A) and TNF-α (FIG. 5B) to GAPDH on days 1, 2 and 7 (n=5 for each)after treatment with control sODN (open bars) or Cx43 asODN (filledbars) are quantified. Data are expressed as the mean±s.e.m. *P<0.05.

FIG. 6A to FIG. 6K depict expression of TGF-β1. FIGS. 6A to 6J depictimmunohistochemistry for TGF-β1 at wound sites treated with control sODN(FIGS. 6A-6E) and with Cx43 asODN (FIGS. 6F-6J). Scale bars represent200 μm (FIGS. 6A and 6F) and 50 μm (FIGS. 6B-6E, and 6G-6J). Blackarrows show the nascent edge of the epidermis. TGF-β1 staining isconsiderably stronger in the epidermis of Cx43 asODN treated wounds(FIGS. 6Iand 6J) compared to control sODN (FIGS. 6D and 6E) wounds.Arrowheads, red and black show representative TGF-β1 elongatedfibroblast-like cells and rounded presumptive leukocytes, respectively.FIG. 6K depicts Real-Time PCR analysis of the expression on days 1, 2and 7 (n=5 for each) of mRNA for TGF-β1 at wound sites treated withcontrol sODN (open bars) or Cx43 asODN (filled bars). Data are expressedas the mean±s.e.m. *P<0.05.

FIG. 7A to FIG. 7F depict granulation tissue formation and fibroblastmigration. FIGS. 7A and 7B depict fibroblast-like cell recruitment intoskin wounds treated with control sODN (FIG. 7A) and Cx43 asODN (FIG.7B), analyzed using TRITC-Phalloidin and DAPI nuclear staining on day 2.FIG. 7C depicts numbers of fibroblast-like cells at each wound site perfield of view for wounds treated with control sODN (open bars: n=5) orCx43 asODN (filled bars: n=5). FIG. 7D depicts results of awound-healing assay of fibroblast migration that shows that migration issignificantly faster after treatment with Cx43 as ODNs. FIGS. 7E and 7Fdepict images of wounds in fibroblast cultures; at the time of wounding(FIG. 7E) and 4 hours after wounding (FIG. 7F). Data are expressed asthe mean±s.e.m. *P<0.02 **P<0.01. Scale bars represent 50 μm.

FIG. 8A to FIG. 8B depict collagen expression in a wound site. FIG. 8Adepicts collagen content assessed by quantitatively measuring thehydroxyproline (HP) content on days 7, 10 and 14 after wounding at woundsites treated with control sODN (open bars) and Cx43 asODN (filled bars)and in uninjured skin (n=5). Data are expressed as the mean±s.e.m.P<0.05. FIG. 8B depicts Real-Time PCR analysis of the expression of mRNAon days 1, 2 and 7 (n=5 for each) for Col1α1 at wound sites treated withcontrol sODN (open bars) and Cx43 asODN (filled bars). Data areexpressed as the mean±s.e.m. *P<0.05.

FIG. 9A to FIG. 9C depict granulation tissue contraction. FIGS. 9A and9B depict H&E staining of 14 days wound granulation tissue in controlsODN treated (FIG. 9A) and asODN treated (FIG. 9B) wounds. FIG. 9Cdepicts an area of granulation tissue after treatment with control sODN(open bars) or Cx43 asODN (filled bars) analyzed on day 5 (control; n=7,asODN; n=6), day 7 (control; n=5, asODN; n=5), day 10 (control; n=5,asODN; n=6), and day 14 (control; n=5, asODN; n=6). Granulation tissuearea measurements at day 5 already showed a slightly smaller area aftertreatment with asODN but the reduction became significant on days 7, 10and 14 (*P<0.05. **P<0.01). Data are expressed as the mean±s.e.m. Scalebars represents 1 mm.

FIG. 10A to FIG. 10C depict apoptosis at wound sites. FIGS. 10A and 10Bdepict TUNEL staining of granulation tissue in control sODN (FIG. 10A)and Cx43 asODN (FIG. 10B) treated wounds on day 7. Apoptotic cellsappear as bright green spots, some of which have been highlighted witharrowheads. Scale bars represents 50 μm. FIG. 10C depicts numbers ofapoptotic cells per field of view on days 5, 7 and 10 (n=6 for each) inwound sites treated with control sODN (open bars) and Cx43 asODN (filedbars). Data are expressed as the mean±s.e.m. Scale bars represent 50 μm.

FIG. 11A to FIG. 11F depict myofibroblast maturation at wound sites.FIGS. 11A and 11D depict anti-α smooth muscle actin (SMA) staining(green) with bis-benzimide nuclear staining (blue) of the edge of thegranulation tissue in a 7 day wound (FIGS. 11A and 11B) and the centerof the granulation tissue in a 10 day wound (FIGS. 11C and 11D). FIG.11E depicts quantification of staining levels showed there to besignificantly more SMA staining in Cx43 asODN treated wounds thancontrols at 7 days (P=0.004) indicating earlier maturation anddifferentiation of myofibroblasts. This more advanced maturation wasstill present at 10 days when most of the SMA staining andmyofibroblasts were lost in Cx43 asODN-treated wounds, but staining wasstill very strong in control wounds (P=0.000002). FIG. 11F depictsillustration of sites imaged in the granulation tissue: zone I (FIGS.11A and 11B) and zone II (FIGS. 11C and 11D). Data are expressed as themean±s.e.m. Scale bars represent 25 μm.

FIG. 12A to FIG. 12H depict angiogenesis at wound sites. FIGS. 12A to12F depict von Willebrand factor staining of granulation tissue nacentblood vessels (green) with bis-benzimide nuclear stain (blue) at 7 days,(FIGS. 12A and 12B) 10 days, (FIGS. 12C and 12D) and 14 days (FIGS. 12Eand 12F) after wounding. In antisense-treated wounds (FIGS. 12A, 12C and12E) blood vessels were more pervasive at early time points (7 daysFIGS. 12A and 12B and 10 days FIGS. 12C and 12D) but considerably finerthan those treated with control ODN (FIGS. 12B, 12D, and 12F) resultingin significantly reduced staining compared to controls (7 days*P=0.0019; 10 days **P=0.015). By 14 days, blood vessels had increasedin size in the asODN group and were a similar size to those of controls(FIG. 12G). FIG. 12H depicts illustration of sites imaged in thegranulation tissue: zone I (FIGS. 12A and 12B) and zone II (FIGS. 12C to12E). Data are expressed as the mean±s. e. m. Scale bars represent 25μm.

FIG. 13A to FIG. 13B depict macroscopic images of wound healing (FIG.13A) and relative changes in the wound area (FIG. 13B) following As ODNtreatment as compared with control treatment.

FIG. 14 depicts a Human Limbal rim—denuded pig stromal matrix chimera.

FIG. 15 depicts examples of growth factor and cytokines binding to theantibody arrays used to detect protein levels for 120 different growthfactors and cytokines. Four samples were run on each of the two arraymembranes (60 growth factors/cytokines per membrane with repeats andboth positive and negative controls).

FIG. 16 depicts analysis of growth factor levels in chimeric stromacompared with limbal rim regions of the chimera after two weeks inculture. This is a control cornea (not antisense treated). Two growthfactors are of particular interest (arrows). These are the very highlevels of IGFBP-2 in the chimeric stroma and higher levels of IGF-1 inthe limbal rim. The former has been reported to be important inpromoting cellular migration (which may be important for the stromalkeratocytes to repopulate the stoma from the limbal rim) and the latterhas been reported to be important in promoting cell proliferation (whichmay be important in the limbal rim to provide the source of cellsrepopulating the stroma).

FIG. 17 depicts analysis of growth factor levels in chimeric stromacompared with limbal rim regions of the chimera after two weeks inculture (based on data from a Cx43As ODN treated cornea). Two growthfactors are of particular interest (arrows). These are the high levelsof IGF-7 in the antisense treated chimeric stroma compared with theuntreated controls and higher levels of IGFBP-2 in both the limbal rimand stroma compared with untreated controls, especially control limbalrims. The former has been reported to be important in promotingepithelial growth (consistent with the increased re-epithelialistaionseen in antisense treated chimeras) and the latter has been reported tobe important in promoting cell migration (consistent with the increasedepithelial repopulation from the limbal rim with antisense treatment).

FIG. 18 illustrates a group of representative cytokines which wereidentified based on their increased levels of expression in aninflammatory compared to low inflammatory wound models. Theserepresentative cytokines can serve as suitable targets for modulation ofwound healing.

FIG. 19 illustrates a group of representative cytokines which wereidentified based on their increased levels of expression in lowinflammatory wound model compared to the inflammatory model. Theserepresentative cytokines can serve as suitable targets for modulation ofwound healing.

DETAILED DESCRIPTION Definitions

As used herein, a “disorder” is any disorder, disease, or condition thatwould benefit from an agent that promotes wound healing and/or reducesswelling, inflammation, and/or scar formation. For example, included arewounds resulting from surgery or trauma, and wound associatedabnormalities in connection with neuropathic, ischemic, microvascularpathology, pressure over bony area (tailbone (sacral), hip(trochanteric), buttocks (ischial), or heel of the foot), reperfusioninjury, and valve reflux etiology and conditions.

As used herein, “subject” refers to any mammal, including humans,domestic and farm animals, and zoo, sports, or pet animals, such asdogs, horses, cats, sheep, pigs, cows, etc. The preferred mammal hereinis a human, including adults, children, and the elderly.

As used herein, “preventing” means preventing in whole or in part, orameliorating or controlling.

As used herein, a “therapeutically effective amount” in reference to thecompounds or compositions of the instant invention refers to the amountsufficient to induce a desired biological, pharmaceutical, ortherapeutic result. That result can be alleviation of the signs,symptoms, or causes of a disease or disorder or condition, or any otherdesired alteration of a biological system. In the present invention, theresult will involve the promotion of wound healing and decreases inswelling, inflammation and/or scar formation in whole or in part.

As used herein, the term “treating” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with a wound or other related disorder as well asthose prone to having a wound or related disorder or diagnosed with thedisorder or those in which the disorder is to be prevented.

As used herein, “anti-connexin agents” are compounds that affect ormodulate the activity, expression or formation of a connexin, a connexinhemichannel (connexon), or a gap junction. Anti-connexin agents include,without limitation, antisense compounds (e.g. antisensepolynucleotides), RNAi and siRNA compounds, antibodies and bindingfragments thereof, and peptides and polypeptides, which include“peptidomimetics,” and peptide analogs. Preferred anti-connexin agentsare anti-connexin 43 agents. Exemplary anit-connexin agents arediscussed in further detail herein.

As used herein, the term “treating” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those prone to havingthe disorder or diagnosed with the disorder or those in which thedisorder is to be prevented.

The terms “peptidomimetic” and “mimetic” include naturally occurring andsynthetic chemical compounds that may have substantially the samestructural and functional characteristics of protein regions which theymimic. In the case of connexins, these may mimic, for example, theextracellular loops of opposing connexins involved in connexon-connexondocking and cell-cell channel formation.

“Peptide analogs” refer to the compounds with properties analogous tothose of the template peptide and may be non-peptide drugs.“Peptidomimetics” (also known as “mimetic peptides”), which includepeptide-based compounds, also include such non-peptide based compoundssuch as peptide analogs. Peptidomimetics that are structurally similarto therapeutically useful peptides may be used to produce an equivalentor enhanced therapeutic or prophylactic effect. Generally,peptidomimetics are structurally identical or similar to a paradigmpolypeptide (i.e., a polypeptide that has a biological orpharmacological function or activity), but can also have one or morepeptide linkages optionally replaced by a linkage selected from thegroup consisting of, for example, —CH2NH—, —CH2S—, —CH2-CH2-, —CH═CH—(cis and trans), —COCH2-, —CH(OH)CH2-, and —CH2SO—. The mimetic can beeither entirely composed of natural amino acids, or non-naturalanalogues of amino acids, or, is a chimeric molecule of partly naturalpeptide amino acids and partly non-natural analogs of amino acids. Themimetic can also comprise any amount of natural amino acid conservativesubstitutions as long as such substitutions also do not substantiallyalter mimetic activity. For example, a mimetic composition may be usefulas an anti-connexin agent if it is capable of down-regulating biologicalactions or activities of connexins proteins or connexons, such as, forexample, preventing the docking of connexons to formgap-junction-mediated cell-cell communications, or preventing theopening connexons to expose the cell cytoplasm to the extracellularmillieu. Peptidomimetics, mimetic peptides, and connexin modulatingpeptides encompass those described such peptidomimetics, mimeticpeptides, and connexin modulating peptides set forth herein, as well asthose as may be known in the art, whether now known or later developed.

The terms “modulator” and “modulation” of connexin activity, as usedherein in its various forms, refers to inhibition in whole or in part ofthe expression or action or activity of a connexin or connexinhemichannel and may function as anti-connexin agents.

In general, the term “protein” refers to any polymer of two or moreindividual amino acids (whether or not naturally occurring) linked viapeptide bonds, as occur when the carboxyl carbon atom of the carboxylicacid group bonded to the alpha-carbon of one amino acid (or amino acidresidue) becomes covalently bound to the amino nitrogen atom of theamino group bonded to the alpha-carbon of an adjacent amino acid. Thesepeptide bond linkages, and the atoms comprising them (i.e., alpha-carbonatoms, carboxyl carbon atoms (and their substituent oxygen atoms), andamino nitrogen atoms (and their substituent hydrogen atoms)) form the“polypeptide backbone” of the protein. In addition, as used herein, theterm “protein” is understood to include the terms “polypeptide” and“peptide” (which, at times, may be used interchangeably herein).Similarly, protein fragments, analogs, derivatives, and variants are maybe referred to herein as “proteins,” and shall be deemed to be a“protein” unless otherwise indicated. The term “fragment” of a proteinrefers to a polypeptide comprising fewer than all of the amino acidresidues of the protein. A “domain” of a protein is also a fragment, andcomprises the amino acid residues of the protein often required toconfer activity or function.

As used herein, “simultaneously” is used to mean that the one or moreagents of the invention are administered concurrently, whereas the term“in combination” is used to mean they are administered, if notsimultaneously or in physical combination, then “sequentially” within atimeframe that they both are available to act therapeutically. Thus,administration “sequentially” may permit one agent to be administeredwithin minutes (for example, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30) minutesor a matter of hours, days, weeks or months after the other providedthat both the one or more anti-connexin agents and one or moretherapeutic agents, agents useful for wound healing, and/or gap junctionmodifying agents are concurrently present in effective amounts. The timedelay between administration or administrations of the components willvary depending on the exact nature of the components, the interactionthere between, and their respective half-lives.

By “wound” is meant an injury to any tissue, including for example,acute, subacute, delayed or difficult to heal wounds, and chronicwounds. Examples of wounds may include both open and closed wounds.Wounds include, for example, burns, incisions, excisions, lacerations,abrasions, puncture or penetrating wounds, surgical wounds, contusions,hematomas, crushing injuries, and ulcers.

As described herein, a delayed or difficult to heal wound may include,for example, a wound that is characterized at least in part by one ormore of 1) a prolonged inflammatory phase, 2) a slow formingextracellular matrix (ECM), and 3) a stalled or decreased rate ofepithelialization.

As used herein, chronic wound may refer to, for example, a wound that ischaracterized at least in part by one or more of 1) a chronicself-perpetuating state of wound inflammation, 2) a deficient anddefective wound ECM, 3) poorly responding (senescent) wound cellsespecially fibroblasts, limiting ECM production, and 4) failure ofre-epithelialization due in part to lack of the necessary ECMorchestration and lack of scaffold for migration. Chronic wounds includevenous ulcers, arterial ulcers, pressure ulcers, vasculitic ulcers, anddiabetic ulcers.

The term “dressing” refers to a dressing for topical application to awound and excludes compositions suitable for systemic administration.For example, the one or more anti-connexin agents and or the one or moreagents useful for wound healing, therapeutic agents, and/or gap junctionmodifying agents may be dispersed in or on a solid sheet of woundcontacting material such as a woven or nonwoven textile material, or maybe dispersed in a layer of foam such as polyurethane foam, or in ahydrogel such as a polyurethane hydrogel, a polyacrylate hydrogel,gelatin, carboxymethyl cellulose, pectin, alginate, and/or hyaluronicacid hydrogel, for example in a gel or ointment. In certain embodimentsthe anti-connexin agent and/or said one or more agents useful for woundhealing, therapeutic agents, and/or gap junction modifying agents aredispersed in or on a biodegradable sheet material that providessustained release of the active ingredients into the wound, for examplea sheet of freeze-dried collagen, freeze-dried collagen/alginatemixtures (available under the Registered Trade Mark FIBRACOL fromJohnson & Johnson Medical Limited) or freeze-dried collagen/oxidizedregenerated cellulose (available under the Registered Trade MarkPROMOGRAN from Johnson & Johnson Medical Limited).

As used herein, “matrix” includes for example, matrices such ascollagen, acellular matrix, crosslinked biological scaffold molecules,tissue based bioengineered structural framework, biomanufacturedbioprostheses, and other implanted structures such as for example,vascular grafts suitable for cell infiltration and proliferation usefulin the promotion of wound healing. Additional suitable biomatrixmaterial may include chemically modified collagenous tissue to reduceantigenicity and immunogenicity. Other suitable examples includecollagen sheets for wound dressings, antigen-free or antigen reducedacellular matrix (Wilson et al., Trans Am Soc Artif Intern 1990;36:340-343) or other biomatrix which have been engineered to reduce theantigenic response to the xenograft material. Other matrix useful inpromotion of wound healing may include for example, processed bovinepericardium proteins comprising insoluble collagen and elastin (Courtmanet al., J Biomed Mater Res 1994; 28:655-666) and other acellular tissuewhich may be useful for providing a natural microenvironment for hostcell migration to accelerate tissue regeneration (Malone et al., J VascSurg 1984; 1:181-91). In certain embodiments, the matrix material may besupplemented with agents useful for wound healing such as growth factorsor other wound healing promoting agents for site specific release,therapeutic agents, and/or gap junction modifying agents.

Wounds and Wound Classification

In addition to the definition previously provided, the term “wound” mayalso include for example, injuries to the skin and subcutaneous tissueinitiated in different ways (e.g., pressure sores from extended bed restand wounds induced by trauma) and with varying characteristics. Woundsmay be classified into one of four grades depending on the depth of thewound: i) Grade I: wounds limited to the epithelium; ii) Grade II:wounds extending into the dermis; iii) Grade III: wounds extending intothe subcutaneous tissue; and iv) Grade IV (or full-thickness wounds):wounds wherein bones are exposed (e.g., a bony pressure point such asthe greater trochanter or the sacrum). The term “partial thicknesswound” refers to wounds that encompass Grades I-III; examples of partialthickness wounds include burn wounds, pressure sores, venous stasisulcers, and diabetic ulcers. The term “deep wound” is meant to includeboth Grade III and Grade IV wounds. The compositions and methods of thepresent invention contemplate treating all wound types, including deepwounds and chronic wounds. The term “chronic wound” refers to a woundthat has not healed. Preferably, it is selected from the groupconsisting of venous ulcers, pressure sores, vasculitic ulcers, diabeticulcers and decubitus ulcers. Chronic skin wounds include, for example,pressure ulcers, diabetic ulcers, venous ulcers, vasculitic ulcers,arterial ulcers, and mixed ulcers. The chronic wound may be an arterialulcer which comprises ulcerations resulting from complete or partialarterial blockage. The chronic wound may be a venous stasis ulcer whichcomprises ulcerations resulting from a malfunction of the venous valveand the associated vascular disease. The chronic wound may be atrauma-induced ulcer, a diabetic ulcer, or a vasculitic ulcer.

Pressure ulcer: Pressure ulcers may be classified into 4 stages based onAHCPR (Agency for Health Care Policy and Research, U.S. Department ofHealth and Human Services) guidelines: Stage 1: A stage I pressure ulceris an observable pressure related alteration of intact skin whoseindicators as compared to the adjacent or opposite area on the body mayinclude changes in one or more of the following: skin temperature(warmth or coolness), tissue consistency (firm or boggy feel) and/orsensation (pain, itching). The ulcer appears as a defined area ofpersistent redness in lightly pigmented skin, whereas in darker skintones, the ulcer may appear with persistent red, blue, or purple hues.Stage 1 ulceration may include nonblanchable erythema of intact skin andthe heralding lesion of skin ulceration. In individuals with darkerskin, discoloration of the skin, warmth, edema, induration, or hardnessmay also be indicators of stage 1 ulceration. Stage 2: stage 2ulceration may be characterized by partial thickness skin loss involvingepidermis, dermis, or both. The ulcer is superficial and presentsclinically as an abrasion, blister, or shallow crater. Stage 3: stage 3ulceration may be characterized by full thickness skin loss involvingdamage to or necrosis of subcutaneous tissue that may extend down to,but not through, underlying fascia. The ulcer presents clinically as adeep crater with or without undermining of adjacent tissue. Stage 4:stage 4 ulceration may be characterized by full thickness skin loss withextensive destruction, tissue necrosis, or damage to muscle, bone, orsupporting structures (e.g., tendon, joint capsule). In certainembodiments compositions and methods of treating a chronic wound areprovided where the chronic wound is characterized by one or more of thefollowing AHCPR stages of pressure ulceration: stage 1, stage 2, stage3, and/or stage 4.

Decubitus ulcers: Decubitus ulcer may arise as a result of prolonged andunrelieved pressure over a bony prominence that leads to ischemia. Thewound tends to occur in patients who are unable to reposition themselvesto off-load weight, such as paralyzed, unconscious, or severelydebilitated persons. As defined by the U.S. Department of Health andHuman Services, the major preventive measures include identification ofhigh-risk patients; frequent assessment; and prophylactic measures suchas scheduled repositioning, appropriate pressure-relief bedding,moisture barriers, and adequate nutritional status. Treatment optionsmay include for example, pressure relief, surgical and enzymaticdebridement, moist wound care, and control of the bacterial load. Incertain embodiments compositions and methods of treating a chronic woundare provided wherein the chronic wound is characterized by decubitusulcer or ulceration which results from prolonged, unrelieved pressureover a bony prominence that leads to ischemia.

Arterial ulcers: Arterial ulcers may be characterized by complete orpartial arterial blockage which may lead to tissue necrosis and/orulceration. Signs of arterial ulcer may include, for example,pulselessness of the extremity; painful ulceration; small, punctateulcers that are usually well circumscribed; cool or cold skin; delayedcapillary return time (briefly push on the end of the toe and release,normal color should return to the toe in about 3 seconds or less);atrophic appearing skin (for example, shiny, thin, dry); and loss ofdigital and pedal hair. In certain embodiments compositions and methodsof treating a chronic wound are provided wherein the chronic wound ischaracterized by arterial ulcers or ulcerations due to complete orpartial arterial blockage.

Venous ulcers: Venous ulcers are the most common type of ulcer affectingthe lower extremities and may be characterized by malfunction of thevenous valve. The normal vein has valves that prevent the backflow ofblood. When these valves become incompetent, the backflow of venousblood causes venous congestion. Hemoglobin from the red blood cellsescapes and leaks into the extravascular space, causing the brownishdiscoloration commonly noted. It has been shown that the transcutaneousoxygen pressure of the skin surrounding a venous ulcer is decreased,suggesting that there are forces obstructing the normal vascularity ofthe area. Lymphatic drainage and flow also plays a role in these ulcers.The venous ulcer may appear near the medial malleolus and usually occursin combination with an edematous and indurated lower extremity; it maybe shallow, not too painful and may present with a weeping dischargefrom the affected site. In certain embodiments compositions and methodsof treating a chronic wound are provided wherein the chronic wound ischaracterized by venous ulcers or ulcerations due to malfunction of thevenous valve and the associated vascular disease.

Venous stasis ulcers: Venous stasis ulcer may be characterized bychronic passive venous congestion of the lower extremities results inlocal hypoxia. One possible mechanism of pathogenesis of these woundsincludes the impediment of oxygen diffusion into the tissue across thickperivascular fibrin cuffs. Another mechanism is that macromoleculesleaking into the perivascular tissue trap growth factors needed for themaintenance of skin integrity. Additionally, the flow of large whiteblood cells slows due to venous congestion, occluding capillaries,becoming activated, and damaging the vascular endothelium to predisposeto ulcer formation. Thus, in certain embodiments compositions and methodof treating a chronic wound are provided wherein the chronic wound ischaracterized by venous stasis ulcers or ulcerations due to chronicpassive venous congestion of the lower extremities and/or the resultinglocal hypoxia.

Diabetic Ulcers: Diabetic patients are prone to foot and otherulcerations due to both neurologic and vascular complications.Peripheral neuropathy can cause altered or complete loss of sensation inthe foot and/or leg. Diabetic patients with advanced neuropathy loosesall ability for sharp-dull discrimination. Any cuts or trauma to thefoot may go completely unnoticed for days or weeks in a patient withneuropathy. A patient with advanced neuropathy looses the ability tosense a sustained pressure insult, as a result, tissue ischemia andnecrosis may occur leading to for example, plantar ulcerations.Microvascular disease is one of the significant complication fordiabetics which may also lead to ulcerations. In certain embodimentscompositions and methods of treating a chronic wound are providedwherein the chronic wound is characterized by diabetic foot ulcersand/or ulcerations due to neurologic and/or vascular complications ofdiabetes.

Traumatic Ulcers: Formation of traumatic ulcers may occur as a result oftraumatic injuries to the body. These injuries include, for example,compromises to the arterial, venous or lymphatic systems; changes to thebony architecture of the skeleton; loss of tissue layers—epidermis,dermis, subcutaneous soft tissue, muscle or bone; damage to body partsor organs and loss of body parts or organs. In certain embodiments,compositions and methods of treating a chronic wound are providedwherein the chronic wound is characterized by ulcerations associatedwith traumatic injuries to the body.

Burn ulcers: Ulceration may also occur as a result of a burn injury,including 1st degree burn (i.e., superficial, reddened area of skin);2nd degree burn (a blistered injury site which may heal spontaneouslyafter the blister fluid has been removed); 3rd degree burn (burn throughthe entire skin and usually require surgical intervention for woundhealing); scalding (may occur from scalding hot water, grease orradiator fluid); thermal (may occur from flames, usually deep burns);chemical (may come from acid and alkali, usually deep burns); electrical(either low voltage around a house or high voltage at work); explosionflash (usually superficial injuries); and contact burns (usually deepand may occur from muffler tail pipes, hot irons and stoves). In certainembodiments, compositions and methods of treating a chronic wound areprovided wherein the chronic wound is characterized by ulcerationsassociated with burn injuries to the body.

Anti-Connexin Agents

Anti-connexin agents of the invention described herein are capable ofmodulating or affecting the transport of molecules into and out of cells(e.g., blocking or inhibiting or downregulating). Thus certainanti-connexin agents described herein modulate cellular communication(e.g., cell to cell). Certain anti-connexin agents modulate or effecttransmission of molecules between the cell cytoplasm and the periplasmicor extracellular space. Such anti-connexin agents are generally targetedto connexins and/or connexin hemichannels (connexons). Hemichannels andresulting gap junctions that comprise connexins are independentlyinvolved in the release or exchange of small molecules between the cellcytoplasm and an extracellular space or tissue in the case of openhemichannels, and between the cytoplasm of adjoining cell in the case ofopen gap junctions. Thus, an anti-connexin agents provided herein maydirectly or indirectly reduce coupling and communication between cellsor reduce or block communication (or the transmission of molecules)between a cell and extracellular space or tissue, and the modulation oftransport of molecules from a cell into an extracellular space or tissue(or from an extracellular space or tissue into a cell) or betweenadjoining cells is within the scope of anti-connexin agents andembodiments of the invention.

Any anti-connexin agent that is capable of eliciting a desiredinhibition of the passage (e.g. transport) of molecules through a gapjunction or connexin hemichannel may be used in embodiments of theinvention. Any anti-connexin agents that modulates the passage ofmolecules through a gap junction or connexin hemichannel are alsoprovided in particular embodiments (e.g., those that modulate, block orlessen the passage of molecules from the cytoplasm of a cell into anextracellular space or adjoining cell cytoplasm). Such anti-connexinagents may modulate the passage of molecules through a gap junction orconnexin hemichannel with or without gap junction uncoupling (blockingthe transport of molecules through gap junctions). Such compoundsinclude, for example, proteins and polypeptides, polynucleotides, andother organic compounds, and they may, for example block the function orexpression of a gap junction or a hemichannel in whole or in part, ordownregulate the production of a connexin in whole or in part. Certaingap junction inhibitors are listed in Evans, W. H. and Boitano, S.Biochem. Soc. Trans. 29: 606-612 (2001).

Certain anti-connexin agents provide downregulation of connexinexpression (for example, by downregulation of mRNA transcription ortranslation) or otherwise decrease or inhibit the activity of a connexinprotein, a connexin hemichannel or a gap junction. In the case ofdownregulation, this will have the effect of reducing direct cell-cellcommunication by gap junctions, or exposure of cell cytoplasm to theextracellular space by hemichannels, at the site at which connexinexpression is downregulated.

Examples of anti-connexin agents include agents that decrease or inhibitexpression or function of connexin mRNA and/or protein or that decreaseactivity, expression or formation of a connexin, a connexin hemichannelor a gap junction. Anti-connexin agents include anti-connexinpolynucleotides, such as antisense polynucleotides and otherpolynucleotides (such as polynucleotides having siRNA or ribozymefunctionalities), as well as antibodies and binding fragments thereof,and peptides and polypeptides, including peptidomimetics and peptideanalogs that modulate hemichannel or gap junction activity or function.

Anti-Connexin Polynucleotides

Anti-connexin polynucleotides include connexin antisense polynucleotidesas well as polynucleotides which have functionalities which enable themto downregulate connexin expression. Other suitable anti-connexinpolynucleotides include RNAi polynucleotides and siRNA polynucleotides.

Synthesis of antisense polynucleotides and other anti-connexinpolynucleotides such as RNAi, siRNA, and ribozyme polynucleotides aswell as polynucleotides having modified and mixed backbones is known tothose of skill in the art. See e.g. Stein C. A. and Krieg A. M. (eds),Applied Antisense Oligonucleotide Technology, 1998 (Wiley-Liss). Methodsof synthesizing antibodies and binding fragments as well as peptides andpolypeptides, including peptidomimetics and peptide analogs are known tothose of skill in the art. See e.g. Lihu Yang et al., Proc. Natl. Acad.Sci. U.S.A., 1; 95(18): 10836-10841 (Sep. 1, 1998); Harlow and Lane(1988) “Antibodies: A Laboratory Manuel” Cold Spring HarborPublications, New York; Harlow and Lane (1999) “Using Antibodies” ALaboratory Manuel, Cold Spring Harbor Publications, New York.

According to one aspect, the downregulation of connexin expression maybe based generally upon the antisense approach using antisensepolynucleotides (such as DNA or RNA polynucleotides), and moreparticularly upon the use of antisense oligodeoxynucleotides (ODN).These polynucleotides (e.g., ODN) target the connexin protein (s) to bedownregulated. Typically the polynucleotides are single stranded, butmay be double stranded.

The antisense polynucleotide may inhibit transcription and/ortranslation of a connexin. Preferably the polynucleotide is a specificinhibitor of transcription and/or translation from the connexin gene ormRNA, and does not inhibit transcription and/or translation from othergenes or mRNAs. The product may bind to the connexin gene or mRNA either(i) 5′ to the coding sequence, and/or (ii) to the coding sequence,and/or (iii) 3′ to the coding sequence.

The antisense polynucleotide is generally antisense to a connexin mRNA.Such a polynucleotide may be capable of hybridizing to the connexin mRNAand may thus inhibit the expression of connexin by interfering with oneor more aspects of connexin mRNA metabolism including transcription,mRNA processing, mRNA transport from the nucleus, translation or mRNAdegradation. The antisense polynucleotide typically hybridizes to theconnexin mRNA to form a duplex which can cause direct inhibition oftranslation and/or destabilization of the mRNA. Such a duplex may besusceptible to degradation by nucleases.

The antisense polynucleotide may hybridize to all or part of theconnexin mRNA. Typically the antisense polynucleotide hybridizes to theribosome binding region or the coding region of the connexin mRNA. Thepolynucleotide may be complementary to all of or a region of theconnexin mRNA. For example, the polynucleotide may be the exactcomplement of all or a part of connexin mRNA. However, absolutecomplementarity is not required and polynucleotides which havesufficient complementarity to form a duplex having a melting temperatureof greater than about 20° C., 30° C. or 40° C. under physiologicalconditions are particularly suitable for use in the present invention.

Thus the polynucleotide is typically a homologue of a sequencecomplementary to the mRNA. The polynucleotide may be a polynucleotidewhich hybridizes to the connexin mRNA under conditions of medium to highstringency such as 0.03M sodium chloride and 0.03M sodium citrate atfrom about 50° C. to about 60° C.

For certain aspects, suitable polynucleotides are typically from about 6to 40 nucleotides in length. Preferably a polynucleotide may be fromabout 12 to about 35 nucleotides in length, or alternatively from about12 to about 20 nucleotides in length or more preferably from about 18 toabout 32 nucleotides in length. According to an alternative aspect, thepolynucleotide may be at least about 40, for example at least about 60or at least about 80, nucleotides in length and up to about 100, about200, about 300, about 400, about 500, about 1000, about 2000 or about3000 or more nucleotides in length.

The connexin protein or proteins targeted by the polynucleotide will bedependent upon the site at which downregulation is to be effected. Thisreflects the non-uniform make-up of gap junction(s) at different sitesthroughout the body in terms of connexin sub-unit composition. Theconnexin is a connexin that naturally occurs in a human or animal in oneaspect or naturally occurs in the tissue in which connexin expression oractivity is to be decreased. The connexin gene (including codingsequence) generally has homology with the coding sequence of one or moreof the specific connexins mentioned herein, such as homology with theconnexin 43 coding sequence shown in Table 8. The connexin is typicallyan α or β connexin. Preferably the connexin is an α connexin and isexpressed in the tissue to be treated.

Some connexin proteins are however more ubiquitous than others in termsof distribution in tissue. One of the most widespread is connexin 43.Polynucleotides targeted to connexin 43 are particularly suitable foruse in the present invention. In other aspects other connexins aretargeted.

Anti-connexin polynucleotides include connexin antisense polynucleotidesas well as polynucleotides which have functionalities which enable themto downregulate connexin expression. Other suitable anti-connexinpolynucleotides include RNAi polynucleotides and SiRNA polynucleotides.

In one preferred aspect, the antisense polynucleotides are targeted tothe mRNA of one connexin protein only. Most preferably, this connexinprotein is connexin 43. In another aspect, connexin protein is connexin26, 30, 31.1, 32, 36, 37, 40, or 45. In other aspects, the connexinprotein is connexin 30.3, 31, 40.1, or 46.6.

It is also contemplated that polynucleotides targeted to separateconnexin proteins be used in combination (for example 1, 2, 3, 4 or moredifferent connexins may be targeted). For example, polynucleotidestargeted to connexin 43, and one or more other members of the connexinfamily (such as connexin 26, 30, 30.3, 31.1, 32, 36, 37, 40, 40.1, 45,and 46.6) can be used in combination.

Alternatively, the antisense polynucleotides may be part of compositionswhich may comprise polynucleotides to more than one connexin protein.Preferably, one of the connexin proteins to which polynucleotides aredirected is connexin 43. Other connexin proteins to whicholigodeoxynucleotides are directed may include, for example, connexins26, 30, 30.3, 31.1, 32, 36, 37, 40, 40.1, 45, and 46.6. Suitableexemplary polynucleotides (and ODNs) directed to various connexins areset forth in Table 1.

Individual antisense polynucleotides may be specific to a particularconnexin, or may target 1, 2, 3 or more different connexins. Specificpolynucleotides will generally target sequences in the connexin gene ormRNA which are not conserved between connexins, whereas non-specificpolynucleotides will target conserved sequences for various connexins.

The polynucleotides for use in the invention may suitably be unmodifiedphosphodiester oligomers. Such oligodeoxynucleotides may vary in length.A 30 mer polynucleotide has been found to be particularly suitable.

Many aspects of the invention are described with reference tooligodeoxynucleotides. However it is understood that other suitablepolynucleotides (such as RNA polynucleotides) may be used in theseaspects.

The antisense polynucleotides may be chemically modified. This mayenhance their resistance to nucleases and may enhance their ability toenter cells. For example, phosphorothioate oligonucleotides may be used.Other deoxynucleotide analogs include methylphosphonates,phosphoramidates, phosphorodithioates, N3′P5′-phosphoramidates andoligoribonucleotide phosphorothioates and their 2′-O-alkyl analogs and2′-O-methylribonucleotide methylphosphonates. Alternatively mixedbackbone oligonucleotides (“MBOs”) may be used. MBOs contain segments ofphosphothioate oligodeoxynucleotides and appropriately placed segmentsof modified oligodeoxy- or oligoribonucleotides. MBOs have segments ofphosphorothioate linkages and other segments of other modifiedoligonucleotides, such as methylphosphonate, which is non-ionic, andvery resistant to nucleases or 2′-O-alkyloligoribonucleotides. Methodsof preparing modified backbone and mixed backbone oligonucleotides areknown in the art.

The precise sequence of the antisense polynucleotide used in theinvention will depend upon the target connexin protein. In oneembodiment, suitable connexin antisense polynucleotides can includepolynucleotides such as oligodeoxynucleotides selected from thefollowing sequences set forth in Table 1:

TABLE 1 5′ GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC 3′ (connexin 43)(SEQ.ID.NO: 1) 5′ GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC 3′(connexin 43) (SEQ.ID.NO: 2) 5′GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT 3′ (connexin 43) (SEQ.ID.NO: 3)5′ TCC TGA GCA ATA CCT AAC GAA CAA ATA 3′ (connexin 26) (SEQ.ID.NO: 4)5′ CAT CTC CTT GGT GCT CAA CC 3′ (connexin 37) (SEQ.ID.NO: 5) 5′CTG AAG TCG ACT TGG CTT GG 3′ (connexin 37) (SEQ.ID.NO: 6) 5′CTC AGA TAG TGG CCA GAA TGC 3′ (connexin 30) (SEQ.ID.NO: 7) 5′TTG TCC AGG TGA CTC CAA GG 3′ (connexin 30) (SEQ.ID.NO: 8) 5′CGT CCG AGC CCA GAA AGA TGA GGT C 3′ (connexin 31.1) (SEQ.ID.NO: 9) 5′AGA GGC GCA CGT GAG ACA C 3′ (connexin 31.1) (SEQ.ID.NO: 10) 5′TGA AGA CAA TGA AGA TGT T 3′ (connexin 31.1) (SEQ.ID.NO: 11) 5′TTT CTT TTC TAT GTG CTG TTG GTG A 3′ (connexin 32) (SEQ.ID.NO: 12)

Suitable polynucleotides for the preparation of the combinedpolynucleotide compositions described herein include for example,polynucleotides to Connexin Cx43 and polynucleotides for connexins 26,30, 31.1, 32 and 37 as described in Table 1 above.

Although the precise sequence of the antisense polynucleotide used inthe invention will depend upon the target connexin protein, for connexin43, antisense polynucleotides having the following sequences have beenfound to be particularly suitable:

(SEQ.ID.NO: 1) GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC; (SEQ.ID.NO: 2)GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC;  and (SEQ.ID.NO: 3)GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT.

For example, suitable antisense polynucleotides for connexins 26, 31.1and 32 have the following sequences:

(connexin 26) (SEQ.ID.NO: 4) 5′ TCC TGA GCA ATA CCT AAC GAA CAA ATA;(connexin 31.1) (SEQ.ID.NO: 9) 5′ CGT CCG AGC CCA GAA AGA TGA GGT C; and (connexin 32) (SEQ.ID.NO: 12) 5′ TTT CTT TTC TAT GTG CTG TTG GTG A.

Other connexin antisense polynucleotide sequences useful according tothe methods of the present invention include:

(connexin 37) (SEQ.ID.NO: 5) 5′ CAT CTC CTT GGT GCT CAA CC 3′;(connexin 37) (SEQ.ID.NO: 6) 5′ CTG AAG TCG ACT TGG CTT GG 3′;(connexin 30) (SEQ.ID.NO: 7) 5′ CTC AGA TAG TGG CCA GAA TGC 3′;(connexin 30) (SEQ.ID.NO: 8) 5′ TTG TCC AGG TGA CTC CAA GG 3′;(connexin 31.1) (SEQ.ID.NO: 10) 5′ AGA GGC GCA CGT GAG ACA C 3′;  and(connexin 31.1) (SEQ.ID.NO: 11) 5′ TGA AGA CAA TGA AGA TGT T 3′.

Polynucleotides, including ODN's, directed to connexin proteins can beselected in terms of their nucleotide sequence by any convenient, andconventional, approach. For example, the computer programs MacVector andOligoTech (from Oligos etc. Eugene, Oreg., USA) can be used. Onceselected, the ODN's can be synthesized using a DNA synthesizer.

Polynucleotide Homologues

Homology and homologues are discussed herein (for example, thepolynucleotide may be a homologue of a complement to a sequence inconnexin mRNA). Such a polynucleotide typically has at least about 70%homology, preferably at least about 80%, at least about 90%, at leastabout 95%, at least about 97% or at least about 99% homology with therelevant sequence, for example over a region of at least about 15, atleast about 20, at least about 40, at least about 100 more contiguousnucleotides (of the homologous sequence).

Homology may be calculated based on any method in the art. For examplethe UWGCG Package provides the BESTFIT program which can be used tocalculate homology (for example used on its default settings) (Devereuxet al (1984) Nucleic Acids Research 12, p 387-395). The PILEUP and BLASTalgorithms can be used to calculate homology or line up sequences(typically on their default settings), for example as described inAltschul S. F. (1993) J Mol Evol 36: 290-300; Altschul, S, F et al(1990) J Mol Biol 215: 403-10.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al, supra). These initial neighborhood wordhits act as seeds for initiating searches to find HSPs containing them.The word hits are extended in both directions along each sequence for asfar as the cumulative alignment score can be increased. Extensions forthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached.

The BLAST algorithm parameters W, T and X determine the sensitivity andspeed of the alignment. The BLAST program uses as defaults a word length(W), the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation(E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a sequenceis considered similar to another sequence if the smallest sumprobability in comparison of the first sequence to a second sequence isless than about 1, preferably less than about 0.1, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The homologous sequence typically differs from the relevant sequence byat least about (or by no more than about) 2, 5, 10, 15, 20 moremutations (which may be substitutions, deletions or insertions). Thesemutations may be measured across any of the regions mentioned above inrelation to calculating homology.

The homologous sequence typically hybridizes selectively to the originalsequence at a level significantly above background. Selectivehybridization is typically achieved using conditions of medium to highstringency (for example 0.03M sodium chloride and 0.03M sodium citrateat from about 50° C. to about 60° C.). However, such hybridization maybe carried out under any suitable conditions known in the art (seeSambrook et al. (1989), Molecular Cloning: A Laboratory Manual). Forexample, if high stringency is required, suitable conditions include0.2×SSC at 60° C. If lower stringency is required, suitable conditionsinclude 2×SSC at 60° C.

Peptide and Polypeptide Anti-Connexin Agents

Binding proteins, including peptides, peptidomimetics, antibodies,antibody fragments, and the like, are also suitable modulators of gapjunctions and hemichannels.

Binding proteins include, for example, monoclonal antibodies, polyclonalantibodies, antibody fragments (including, for example, Fab, F(ab′)₂ andFv fragments; single chain antibodies; single chain Fvs; and singlechain binding molecules such as those comprising, for example, a bindingdomain, hinge, CH2 and CH3 domains, recombinant antibodies and antibodyfragments which are capable of binding an antigenic determinant (i.e.,that portion of a molecule, generally referred to as an epitope) thatmakes contact with a particular antibody or other binding molecule.These binding proteins, including antibodies, antibody fragments, and soon, may be chimeric or humanized or otherwise made to be lessimmunogenic in the subject to whom they are to be administered, and maybe synthesized, produced recombinantly, or produced in expressionlibraries. Any binding molecule known in the art or later discovered isenvisioned, such as those referenced herein and/or described in greaterdetail in the art. For example, binding proteins include not onlyantibodies, and the like, but also ligands, receptors, peptidomimetics,or other binding fragments or molecules (for example, produced by phagedisplay) that bind to a target (e.g. connexin, hemichannel, orassociated molecules).

Binding molecules will generally have a desired specificity, includingbut not limited to binding specificity, and desired affinity. Affinity,for example, may be a K_(a) of greater than or equal to about 10⁴ M⁻¹,greater than or equal to about 10⁶ M⁻¹, greater than or equal to about10⁷ M⁻¹, greater than or equal to about 10⁸ M⁻¹. Affinities of evengreater than about 10⁸ M⁻¹ are suitable, such as affinities equal to orgreater than about 10⁹ M⁻¹, about 10¹⁰ M⁻¹, about 10¹¹ M⁻¹, and about10¹² M⁻¹. Affinities of binding proteins according to the presentinvention can be readily determined using conventional techniques, forexample those described by Scatchard et al., 1949 Ann. N.Y. Acad. Sci.51: 660.

By using data obtained from hydropathy plots, it has been proposed thata connexin contains four-transmembrane-spanning regions and two shortextra-cellular loops. The positioning of the first and secondextracellular regions of connexin was further characterized by thereported production of anti-peptide antibodies used forimmunolocalization of the corresponding epitopes on split gap junctions.Goodenough D. A. J Cell Biol 107: 1817-1824 (1988); Meyer R. A., J CellBiol 119: 179-189 (1992).

The extracellular domains of a hemichannel contributed by two adjacentcells “dock” with each other to form complete gap junction channels.Reagents that interfere with the interactions of these extracellulardomains can impair cell-to-cell communication. Peptide inhibitors of gapjunctions and hemichannels have been reported. See for example Berthoud,V. M. et al., Am J. Physiol. Lung Cell Mol. Physiol. 279: L619-L622(2000); Evans, W. H. and Boitano, S. Biochem. Soc. Trans. 29: 606-612,and De Vriese A. S., et al. Kidney Int. 61: 177-185 (2001). Shortpeptides corresponding to sequences within the extracellular loops ofconnexins were said to inhibit intercellular communication. Boitano S.and Evans W. Am J Physiol Lung CellMol Physiol 279: L623-L630 (2000).The use of peptides as inhibitors of cell-cell channel formationproduced by connexin (Cx) 32 expressed in paired Xenopus oocytes hasalso been reported. Dahl G, et al., Biophys J 67: 1816-1822 (1994).Berthoud, V. M. and Seul, K. H., summarized some of these results. AmJ., Physiol. Lung Cell Mol. Physiol. 279: L619-L622 (2000).

Anti-connexin agents include peptides comprising an amino acid sequencecorresponding to a transmembrane region (e.g. 1^(st) to 4^(th)) of aconnexin (e.g. connexin 45, 43, 26, 30, 31.1, and 37). Anti-connexinagents may comprise a peptide comprising an amino acid sequencecorresponding to a portion of a transmembrane region of a connexin 45.Anti-connexin agents include a peptide having an amino acid sequencethat comprises about 5 to 20 contiguous amino acids of SEQ.ID.NO:13, apeptide having an amino acid sequence that comprises about 8 to 15contiguous amino acids of SEQ.ID.NO: 13, or a peptide having an aminoacid sequence that comprises about 11 to 13 contiguous amino acids ofSEQ.ID.NO:13. Other embodiments are directed to an anti-connexin agentthat is a peptide having an amino acid sequence that comprises at leastabout 5, at least about 6, at least about 7, at least about 8, at leastabout 9, at least about 10, at least about 11, at least about 12, atleast about 13, at least about 14, at least about 15, at least about 20,at least about 25, or at least about 30 contiguous amino acids ofSEQ.ID.NO:13. In certain anti-connexin agents provided herein, theextracellular domains of connexin 45 corresponding to the amino acids atpositions 46-75 and 199-228 of SEQ ID NO: 13 may be used to develop theparticular peptide sequences. Certain peptides described herein have anamino acid sequence corresponding to the regions at positions 46-75 and199-228 of SEQ.ID.NO: 13. The peptides need not have an amino acidsequence identical to those portions of SEQ.ID.NO: 13, and conservativeamino acid changes may be made such that the peptides retain bindingactivity or functional activity. Alternatively, the peptide may targetregions of the connexin protein other than the extracellular domains(e.g. the portions of SEQ.ID.NO:13 not corresponding to positions 46-75and 199-228).

Also, suitable anti-connexin agents comprise a peptide comprising anamino acid sequence corresponding to a portion of a transmembrane regionof a connexin 43. Anti-connexin agents include peptides having an aminoacid sequence that comprises about 5 to 20 contiguous amino acids ofSEQ.ID.NO: 14, peptides having an amino acid sequence that comprisesabout 8 to 15 contiguous amino acids of SEQ.ID.NO:14, or peptides havingan amino acid sequence that comprises about 11 to 13 contiguous aminoacids of SEQ.ID.NO: 14. Other anti-connexin agents include a peptidehaving an amino acid sequence that comprises at least about 5, at leastabout 6, at least about 7, at least about 8, at least about 9, at leastabout 10, at least about 11, at least about 12, at least about 13, atleast about 14, at least about 15, at least about 20, at least about 25,or at least about 30 contiguous amino acids of SEQ.ID.NO:14. Otheranti-connexin agents comprise the extracellular domains of connexin 43corresponding to the amino acids at positions 37-76 and 178-208 ofSEQ.ID.NO: 14. Anti-connexin agents include peptides described hereinwhich have an amino acid sequence corresponding to the regions atpositions 37-76 and 178-208 of SEQ.ID.NO: 14. The peptides need not havean amino acid sequence identical to those portions of SEQ.ID.NO: 14, andconservative amino acid changes may be made such that the peptidesretain binding activity or functional activity. Alternatively, peptidesmay target regions of the connexin protein other than the extracellulardomains (e.g. the portions of SEQ.ID.NO:14 not corresponding topositions 37-76 and 178-208).

Connexin 45 (SEQ ID NO. 13)Met Ser Trp Ser Phe Leu Thr Arg Leu Leu Glu Glu Ile His Asn His1               5                   10                  15Ser Thr Phe Val Gly Lys Ile Trp Leu Thr Val Leu Ile Val Phe Arg            20                  25                  30Ile Val Leu Thr Ala Val Gly Gly Glu Ser Ile Tyr Tyr Asp Glu Gln        35                  40                  45Ser Lys Phe Val Cys Asn Thr Glu Gln Pro Gly Cys Glu Asn Val Cys    50                  55                  60Tyr Asp Ala Phe Ala Pro Leu Ser His Val Arg Phe Trp Val Phe Gln65                  70                  75                  80Ile Ile Leu Val Ala Thr Pro Ser Val Met Tyr Leu Gly Tyr Ala Ile                85                  90                  95His Lys Ile Ala Lys Met Glu His Gly Glu Ala Asp Lys Lys Ala Ala            100                 105                 110Arg Ser Lys Pro Tyr Ala Met Arg Trp Lys Gln His Arg Ala Leu Glu        115                 120                 125Glu Thr Glu Glu Asp Asn Glu Glu Asp Pro Met Met Tyr Pro Glu Met    130                 135                 140Glu Leu Glu Ser Asp Lys Glu Asn Lys Glu Gln Ser Gln Pro Lys Pro145                 150                 155                 160Lys His Asp Gly Arg Arg Arg Ile Arg Glu Asp Gly Leu Met Lys Ile                165                 170                 175Tyr Val Leu Gln Leu Leu Ala Arg Thr Val Phe Glu Val Gly Phe Leu            180                 185                 190Ile Gly Gln Tyr Phe Leu Tyr Gly Phe Gln Val His Pro Phe Tyr Val        195                 200                 205Cys Ser Arg Leu Pro Cys Pro His Lys Ile Asp Cys Phe Ile Ser Arg    210                 215                 220Pro Thr Glu Lys Thr Ile Phe Leu Leu Ile Met Tyr Gly Val Thr Gly225                 230                 235                 240Leu Cys Leu Leu Leu Asn Ile Trp Glu Met Leu His Leu Gly Phe Gly                245                 250                 255Thr Ile Arg Asp Ser Leu Asn Ser Lys Arg Arg Glu Leu Glu Asp Pro            260                 265                 270Gly Ala Tyr Asn Tyr Pro Phe Thr Trp Asn Thr Pro Ser Ala Pro Pro        275                 280                 285Gly Tyr Asn Ile Ala Val Lys Pro Asp Gln Ile Gln Tyr Thr Glu Leu    290                 295                 300Ser Asn Ala Lys Ile Ala Tyr Lys Gln Asn Lys Ala Asn Thr Ala Gln305                 310                 315                 320Glu Gln Gln Tyr Gly Ser His Glu Glu Asn Leu Pro Ala Asp Leu Glu                325                 330                 335Ala Leu Gln Arg Glu Ile Arg Met Ala Gln Glu Arg Leu Asp Leu Ala            340                 345                 350Val Gln Ala Tyr Ser His Gln Asn Asn Pro His Gly Pro Arg Glu Lys        355                 360                 365Lys Ala Lys Val Gly Ser Lys Ala Gly Ser Asn Lys Ser Thr Ala Ser    370                 375                 380Ser Lys Ser Gly Asp Gly Lys Asn Ser Val Trp Ile385                 390                 395 Connexin 43 (SEQ ID NO. 14)Met Gly Asp Trp Ser Ala Leu Gly Lys Leu Leu Asp Lys Val Gln Ala1               5                   10                  15Tyr Ser Thr Ala Gly Gly Lys Val Trp Leu Ser Val Leu Phe Ile Phe            20                  25                  30Arg Ile Leu Leu Leu Gly Thr Ala Val Glu Ser Ala Trp Gly Asp Glu        35                  40                  45Gln Ser Ala Phe Arg Cys Asn Thr Gln Gln Pro Gly Cys Glu Asn Val    50                  55                  60Cys Tyr Asp Lys Ser Phe Pro Ile Ser His Val Arg Phe Trp Val Leu65                  70                  75                  80Gln Ile Ile Phe Val Ser Val Pro Thr Leu Leu Tyr Leu Ala His Val                85                  90                  95Phe Tyr Val Met Arg Lys Glu Glu Lys Leu Asn Lys Lys Glu Glu Glu            100                 105                 110Leu Lys Val Ala Gln Thr Asp Gly Val Asn Val Asp Met His Leu Lys        115                 120                 125Gln Ile Glu Ile Lys Lys Phe Lys Tyr Gly Ile Glu Glu His Gly Lys    130                 135                 140Val Lys Met Arg Gly Gly Leu Leu Arg Thr Tyr Ile Ile Ser Ile Leu145                 150                 155                 160Phe Lys Ser Ile Phe Glu Val Ala Phe Leu Leu Ile Gln Trp Tyr Ile                165                 170                 175Tyr Gly Phe Ser Leu Ser Ala Val Tyr Thr Cys Lys Arg Asp Pro Cys            180                 185                 190Pro His Gln Val Asp Cys Phe Leu Ser Arg Pro Thr Glu Lys Thr Ile        195                 200                 205Phe Ile Ile Phe Met Leu Val Val Ser Leu Val Ser Leu Ala Leu Asn    210                 215                 220Ile Ile Glu Leu Phe Tyr Val Phe Phe Lys Gly Val Lys Asp Arg Val225                 230                 235                 240Lys Gly Lys Ser Asp Pro Tyr His Ala Thr Ser Gly Ala Leu Ser Pro                245                 250                 255Ala Lys Asp Cys Gly Ser Gln Lys Tyr Ala Tyr Phe Asn Gly Cys Ser            260                 265                 270Ser Pro Thr Ala Pro Leu Ser Pro Met Ser Pro Pro Gly Tyr Lys Leu        275                 280                 285Val Thr Gly Asp Arg Asn Asn Ser Ser Cys Arg Asn Tyr Asn Lys Gln    290                 295                 300Ala Ser Glu Gln Asn Trp Ala Asn Tyr Ser Ala Glu Gln Asn Arg Met305                 310                 315                 320Gly Gln Ala Gly Ser Thr Ile Ser Asn Ser His Ala Gln Pro Phe Asp                325                 330                 335Phe Pro Asp Asp Asn Gln Asn Ser Lys Lys Leu Ala Ala Gly His Glu            340                 345                 350Leu Gln Pro Leu Ala Ile Val Asp Gln Arg Pro Ser Ser Arg Ala Ser        355                 360                 365Ser Arg Ala Ser Ser Arg Pro Arg Pro Asp Asp Leu Glu Ile    370                 375                 380

The anti-connexin peptides may comprise sequences corresponding to aportion of the connexin extracellular domains with conservative aminoacid substitutions such that peptides are functionally activeanti-connexin agents. Exemplary conservative amino acid substitutionsinclude for example the substitution of a nonpolar amino acid withanother nonpolar amino acid, the substitution of an aromatic amino acidwith another aromatic amino acid, the substitution of an aliphatic aminoacid with another aliphatic amino acid, the substitution of a polaramino acid with another polar amino acid, the substitution of an acidicamino acid with another acidic amino acid, the substitution of a basicamino acid with another basic amino acid, and the substitution of anionizable amino acid with another ionizable amino acid.

Exemplary peptides targeted to connexin 43 are shown below in Table 2.M1, 2, 3 and 4 refer to the 1^(st) to 4^(th) transmembrane regions ofthe connexin 43 protein respectively. E1 and E2 refer to the first andsecond extracellular loops respectively.

TABLE 2 Peptidic Inhibitors of Intercellular Communication (cx43)FEVAFLLIQWI M3 & E2 (SEQ.ID.NO: 15) LLIQWYIGFSL E2 (SEQ.ID.NO: 16)SLSAVYTCKRDPCPHQ E2 (SEQ.ID.NO: 17) VDCFLSRPTEKT E2 (SEQ.ID.NO: 18)SRPTEKTIFII E2 & M4 (SEQ.ID.NO: 19) LGTAVESAWGDEQ M1 & E1(SEQ.ID.NO: 20) QSAFRCNTQQPG E1 (SEQ.ID.NO: 21) QQPGCENVCYDK E1(SEQ.ID.NO: 22) VCYDKSFPISHVR E1 (SEQ.ID.NO: 23)

Table 3 provides additional exemplary connexin peptides used ininhibiting hemichannel or gap junction function. In other embodiments,conservative amino acid changes are made to the peptides or fragmentsthereof.

TABLE 3Additional Peptidic Inhibitors of Intercellular Communication (cx32, cx43)AA′s and Connexin Location Sequence Cx32 E1 39-77AAESVWGDEIKSSFICNTLQPGCNSVCYDHFFPIS (SEQ.ID.NO: 24) HVR Cx32 E1 41-52ESVWGDEKSSFI (SEQ.ID.NO: 25) Cx32 E1 52-63 ICNTLQPGCNSV (SEQ.ID.NO: 26)Cx32 E1 62-73 SVCYDHFFPISH (SEQ.ID.NO: 27) Cx32 E2 64-188RLVKCEAFPCPNTVDCFVSRPTEKT (SEQ.ID.NO: 28) Cx32 E2 166-177 VKCEAFPCPNTV(SEQ.ID.NO: 29) Cx32 E1 177-188 VDCFVSRPTEKT (SEQ.ID.NO: 30) Cx32E1 63-75 VCYDHFFPISHVR (SEQ.ID.NO: 31) Cx32 E1 45-59 VWGDEKSSFICNTLQPGY(SEQ.ID.NO: 32) Cx32 E1 46-59 DEKSSFICNTLQPGY (SEQ.ID.NO: 33) Cx32E2 182-192 SRPTEKTVFTV (SEQ.ID.NO: 34) Cx32/Cx43 E2 182-188/ SRPTEKT(SEQ.ID.NO: 35) 201-207 Cx32 E1 52-63 ICNTLQPGCNSV (SEQ.ID.NO: 36) Cx40E2 177-192 FLDTLHVCRRSPCPHP (SEQ.ID.NO: 37) Cx43 E2 188-205KRDPCHQVDCFLSRPTEK (SEQ.ID.NO: 38)

Table 4 provides the extracellular loops for connexin family memberswhich are used to develop peptide inhibitors for use as describedherein. The peptides and provided in Table 4, and fragments thereof, areused as peptide inhibitors in certain non-limiting embodiments. In othernon-limiting embodiments, peptides comprising from about 8 to about 15,or from about 11 to about 13 amino contiguous amino acids of thepeptides in this Table 4 are peptide inhibitors. Conservative amino acidchanges may be made to the peptides or fragments thereof.

TABLE 4 Extracellular loops for various connexin family members E1huCx26 KEVWGDEQADFVCNTLQPGCKNVCYDHYFPISHIR (SEQ.ID.NO: 39) huCx30QEVWGDEQEDFVCNTLQPGCKNVCYDHFFPVSHIR (SEQ.ID.NO: 40) huCx30.3EEVWDDEQKDFVCNTKQPGCPNVCYDEFFPVSHVR (SEQ.ID.NO: 41) huCx31ERVWGDEQKDFDCNTKQPGCTNVCYDNYFPISNIR (SEQ.ID.NO: 42) huCx31.1ERVWSDDHKDFDCNTRQPGCSNVCFDEFFPVSHVR (SEQ.ID.NO: 43) huCx32ESVWGDEKSSFICNTLQPGCNSVCYDQFFPISHVR (SEQ.ID.NO: 44) huCx36ESVWGDEQSDFECNTAQPGCTNVCYDQAFPISHIR (SEQ.ID.NO: 45) huCx37ESVWGDEQSDFECNTAQPGCTNVCYDQAFPISHIR (SEQ.ID.NO: 46) huCx40.1RPVYQDEQERFVCNTLQPGCANVCYDVFSPVSHLR (SEQ.ID.NO: 47) huCx43ESAWGDEQSAFRCNTQQPGCENVCYDKSFPISHVR (SEQ.ID.NO: 48) huCx46EDVWGDEQSDFTCNTQQPGCBNVCYBRAFPISHIR (SEQ.ID.NO: 49) huCx46.6EAIYSDEQAKFTCNTRQPGCDNVCYDAFAPLSHVR (SEQ.ID.NO: 50) huCx40ESSWGDEQADFRCDTIQPGCQNVCTDQAFPISHIR (SEQ.ID.NO: 51) huCx45GESIYYDEQSKFVCNTEQPGCENVCYDAFAPLSHVR (SEQ.ID NO: 52) E2 huCx26MYVFYVMYDGFSMQRLVKCNAWPCPNTVDCFVSRPTEKT (SEQ.ID.NO: 53) huCx30MYVFYFLYNGYHLPWVLKCGIDPCPNLVDCFISRPTEKT (SEQ.ID.NO: 54) huCx30.3LYIFHRLYKDYDMPRVVACSVEPCPHTVDCYISRPTEKK (SEQ.ID.NO: 55) huCx31LYLLHTLWHGFNMPRLVQCANVAPCPNIVDCYIARPTEKK (SEQ.ID.NO: 56) huCx31.1LYVFHSFYPKYILPPVVKCHADPCPNIVDCFISKPSEKN (SEQ.ID.NO: 57) huCx32MYVFYLLYPGYAMVRLVKCDVYPCPNTVDCFVSRPTEKT (SEQ.ID.NO: 58) huCx36LYGWTMEPVFVCQRAPCPYLVDCFVSRPTEKT (SEQ.ID.NO: 59) huCx37LYGWTMEPVFVCQRAPCPYLVDCFVSRPTEKT (SEQ.ID.NO: 60) huCx40.1GALHYFLFGFLAPKKFPCTRPPCTGVVDCYVSRPTSKS (SEQ.ID.NO: 61) huCx43LLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKT (SEQ.ID NO: 62) huCx46IAGQYFLYGFELKPLYRCDRWPCPNTVDCFISRPTEKT (SEQ.ID.NO: 63) huCx46.6LVGQYLLYGFEVRPFFPCSRQPCPHVVDCFVSRPTEKT (SEQ.ID NO: 64) huCx40IVGQYFIYGIFLTTLHVCRRSPCPHPVNCYVSRPTEKN (SEQ.ID NO: 65) huCx45LIGQYFLYGFQVHPFYVCSRLPCHPKIDCFISRPTEKT (SEQ.ID.NO: 66)

Table 5 provides the extracellular domain for connexin family memberswhich may be used to develop peptide anti-connexin agents. The peptidesand provided in Table 5, and fragments thereof, may also be used aspeptide anti-connexin agents. Such peptides may comprise from about 8 toabout 15, or from about 11 to about 13 amino contiguous amino acids ofthe peptide sequence in this Table 5. Conservative amino acid changesmay be made to the peptides or fragments thereof.

TABLE 5 Extracellular domains Peptide VDCFLSRPTEKT (SEQ.ID.NO: 18)Peptide SRPTEKTIFII (SEQ.ID.NO: 19) huCx43LLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTIFII (SEQ.ID.NO: 67) huCx26MYVFYVMYDGFSMQRLVKCNAWPCPNTVDCFVSRPTEKTVFTV (SEQ.ID.NO: 68) huCx30YVFYFLYNGYHLPWVLKCGIDPCPNLVDCFISRPTEKTVFTI (SEQ.ID.NO: 69) huCx30.3LYIFHRLYKDYDMPRVVACSVEPCPHTVDCYISRPTEKKVFTY (SEQ.ID.NO: 70) huCx31LYLLHTLWHGFNMPRLVQCANVAPCPNIVDCYIARPTEKKTY (SEQ.ID.NO: 71) huCx31.1LYVFHSFYPKYILPPVVKCHADPCPNIVDCFISKPSEKNIFTL (SEQ.ID.NO: 72) huCx32MYVFYLLYPGYAMVRLVKCDVYPCPNTVDCFVSRPTEKTVFTV (SEQ.ID.NO: 73) huCx36LYGWTMEPVFVCQRAPCPYLVDCFVSRPTEKTIFII (SEQ.ID.NO: 74) huCx37LYGWTMEPVFVCQRAPCPYLVDCFVSRPTEKTIFII (SEQ.ID.NO: 75) huCx40.1GALHYFLFGFLAPKKFPCTRPPCTGVVDCYVSRPTEKSLLML (SEQ.ID.NO: 76) huCx46IAGQYFLYGFELKPLYRCDRWPCPNTVDCFISRPTEKTIFII (SEQ.ID.NO: 77) huCx46.6LVGQYLLYGFEVRPFFPCSRQPCPHVVDCFVSRPTEKTVFLL (SEQ.ID.NO: 78) huCx40IVGQYFIYGIFLTTLHVCRRSPCPHPVNCYSRPTEKNVFIV (SEQ.ID.NO: 79) huCx45LIGQYFLYGFQVHPFYVCSRLPCHPKIDCFISRPTEKTIFLL (SEQ.ID.NO: 80)

Table 6 provides peptides inhibitors of connexin 40 shown with referenceto the extracellular loops (E1 and E2) of connexin 40. The bold aminoacids are directed to the transmembrane regions of connexin 40.

TABLE 6 Cx40 peptide inhibitors E2LGTAAESSWGDEQADFRCDTIQPGCQNVCIDQAFPISHIRFWVLQ (SEQ.ID.NO: 94)LGTAAESSWGDEQA (SEQ.ID.NO: 94)           DEQADFRCDTIQP (SEQ.ID.NO: 94)                   TIQPGCQNVCTDQ (SEQ.ID.NO: 94)                           VCTDQAFPISHIR (SEQ.ID.NO: 94)                                AFPISHIRFWVLQ (SEQ.ID.NO: 94) E2MEVGFIVGQYFIYGIFLTTLHVCRRSPCPHPVNCYVSRPTEKNVFIV (SEQ.ID.NO: 94)MEVGFIVGQYF (SEQ.ID.NO: 94)      IVGQYFIYGIFL (SEQ.ID.NO: 94)             GIFLTTLHVCRRSP (SEQ.ID.NO: 94)                       RRSPCPHPVNCY (SEQ.ID.NO: 94)                               VNCYVSRPTEKN (SEQ.ID.NO: 94)                                    SRPTEKNVFIV (SEQ.ID.NO: 94)

Table 7 provides peptides inhibitors of connexin 45 shown with referenceto the extracellular loops (E1 and E2) of connexin 45. The bold aminoacids are directed to the transmembrane regions of connexin 45

TABLE 7 Cx45 peptide inhibitors E1 LTAVGGESIYYDEQSKFVCNTEQPGCENVCYDAFAPLSHVRFWVFQ (SEQ.ID.NO: 94)LTAVGGESIYYDEQS (SEQ.ID.NO: 95)            DEQSKFVCNTEQP (SEQ.ID.NO: 96)                    TEQPGCENVCYDA (SEQ.ID.NO: 97)                            VCYDAFAPLSHVR (SEQ.ID.NO: 98)                                  APLSHVRFWVFQ (SEQ.ID.NO: 99) E2FEVGFLIGQYFLYGFQVHPFYVCSRLPCHPKIDCFISRPTEKTIFLL (SEQ.ID.NO: 100)FEVGFLIGQYF (SEQ.ID.NO: 101)      LIGQYFLYGFQV (SEQ.ID.NO: 102)             GFQVHPFYVCSRLP (SEQ.ID.NO: 103)                       SRLPCHPKIDCF (SEQ.ID.NO: 104)                               IDCFISRPTEKT (SEQ.ID.NO: 105)                                    SRPTEKTIFLL (SEQ.ID.NO: 106)

In certain embodiments, it is preferred that certain peptide inhibitorsblock hemichannels without disrupting existing gap junctions. While notwishing to be bound to any particular theory or mechanism, it is alsobelieved that certain peptidomimetics (e.g. VCYDKSFPISHVR, (SEQ.ID.NO:23) block hemichannels without causing uncoupling of gap junctions (SeeLeybeart et al., Cell Commun. Adhes. 10: 251-257 (2003)), or do so inlower dose amounts. The peptide SRPTEKTIFII (SEQ.ID.NO: 19) may also beused, for example to block hemichannels without uncoupling of gapjunctions. The peptide SRGGEKNVFIV (SEQ.ID.NO: 107) may be used that asa control sequence (DeVriese et al., Kidney Internat. 61: 177-185(2002)). Examples of peptide inhibitors for connexin 45 YVCSRLPCHP(SEQ.ID.NO:108), QVHPFYVCSRL (SEQ.ID.NO: 109), FEVGFLIGQYFLY (SEQ.ID.NO:110), GQYFLYGFQVHP (SEQ.ID.NO: 111), GFQVHPFYVCSR (SEQ.ID.NO: 112),AVGGESIYYDEQ (SEQ.ID.NO), YDEQSKFVCNTE (SEQ.ID.NO:114), NTEQPGCENVCY(SEQ.ID.NO:115), CYDAFAPLSHVR (SEQ.ID.NO: 116), FAPLSHVRFWVF(SEQ.ID.NO:117) and LIGQY (SEQ.ID.NO:118), QVHPF (SEQ.ID.NO:119), YVCSR(SEQ.ID.NO:120), SRLPC (SEQ.ID.NO:121), LPCHP (SEQ.ID.NO:122) and GESIY(SEQ.ID.NO:123), YDEQSK (SEQ.ID.NO:124), SKFVCN (SEQ.ID.NO:125),TEQPGCEN (SEQ.ID.NO:126), VCYDAFAP (SEQ.ID.NO:127), LSHVRFWVFQ(SEQ.ID.NO:128) The peptides may only be 3 amino acids in length,including SRL, PCH, LCP, CHP, IYY, SKF, QPC, VCY, APL, HVR, or longer,for example: LIQYFLYGFQVHPF (SEQ.ID.NO:129), VHPFYCSRLPCHP(SEQ.ID.NO:130), VGGESIYYDEQSKFVCNTEQPG (SEQ.ID.NO:131),TEQPGCENVCYDAFAPLSHVRF (SEQ.ID.NO:132), AFAPLSHVRFWVFQ (SEQ.ID.NO: 133).

TABLE 8 Table 8AHuman Connexin 43 from GenBank Accession No. M65188 (SEQ.ID.NO: 134) 1ggcttttagc gtgaggaaag taccaaacag cagcggagtt ttaaacttta aatagacagg 61tctgagtgcc tgaacttgcc ttttcattft acttcatcct ccaaggagtt caatcacttg 121gcgtgacttc actactttta agcaaaagag tggtgcccag gcaacatggg tgactggagc 181gccttaggca aactccttga caaggttcaa gcctactcaa ctgctggagg gaaggtgtgg 241ctgtcagtac ttttcattft ccgaatcctg ctgctgggga cagcggttga gtcagcctgg 301ggagatgagc agtctgcctt tcgttgtaac actcagcaac ctggttgtga aaatgtctgc 361tatgacaagt ctttcccaat ctctcatgtg cgcttctggg tcctgcagat catatttgtg 421tctgtaccca cactcttgta cctggctcat gtgttctatg tgatgcgaaa ggaagagaaa 481ctgaacaaga aagaggaaga actcaaggtt gcccaaactg atggtgtcaa tgtggacatg 541cacttgaagc agattgagat aaagaagttc aagtacggta ttgaagagca tggtaaggtg 601aaaatgcgag gggggttgct gcgaacctac atcatcagta tcctcttcaa gtctatcttt 661gaggtggcct tcttgctgat ccagtggtac atctatggat tcagcttgag tgctgtttac 721acttgcaaaa gagatccctg cccacatcag gtggactgtt tcctctctcg ccccacggag 781aaaaccatct tcatcatctt catgctggtg gtgtccttgg tgtccctggc cttgaatatc 841attgaactct tctatgtttt cttcaagggc gttaaggatc gggttaaggg aaagagcgac 901ccttaccatg cgaccagtgg tgcgctgagc cctgccaaag actgtgggtc tcaaaaatat 961gcttatttca atggctgctc ctcaccaacc gctcccctct cgcctatgtc tcctcctggg 1021tacaagctgg ttactggcga cagaaacaat tcttcttgcc gcaattacaa caagcaagca 1081agtgagcaaa actgggctaa ttacagtgca gaacaaaatc gaatggggca ggcgggaagc 1141accatctcta actcccatgc acagcctttt gatttccccg atgataacca gaattctaaa 1201aaactagctg ctggacatga attacagcca ctagccattg tggaccagcg accttcaagc 1261agagccagca gtcgtgccag cagcagacct cggcctgatg acctggagat ctag Table 8BHuman Connexin43 (SEQ.ID.NO: 135) 1atgggtgact ggagcgcctt aggcaaactc cttgacaagg ttcaagccta ctcaactgct 61ggagggaagg tgtggctgtc agtacttttc attttccgaa tcctgctgct ggggacagcg 121gttgagtcag cctggggaga tgagcagtct gcctttcgtt gtaacactca gcaacctggt 181tgtgaaaatg tctgctatga caagtctttc ccaatctctc atgtgcgctt ctgggtcctg 241cagatcatat ttgtgtctgt acccacactc ttgtacctgg ctcatgtgtt ctatgtgatg 301cgaaaggaag agaaactgaa caagaaagag gaagaactca aggttgccca aactgatggt 361gtcaatgtgg acatgcactt gaagcagatt gagataaaga agttcaagta cggtattgaa 421gagcatggta aggtgaaaat gcgagggggg ttgctgcgaa cctacatcat cagtatcctc 481ttcaagtcta tctttgaggt ggccttcttg ctgatccagt ggtacatcta tggattcagc 541ttgagtgctg tttacacttg caaaagagat ccctgcccac atcaggtgga ctgtttcctc 601tctcgcccca cggagaaaac catcttcatc atcttcatgc tggtggtgtc cttggtgtcc 661ctggccttga atatcattga actcttctat gttttcttca agggcgttaa ggatcgggtt 721aagggaaaga gcgaccctta ccatgcgacc agtggtgcgc tgagccctgc caaagactgt 781gggtctcaaa aatatgctta tttcaatggc tgctcctcac caaccgctcc cctctcgcct 841atgtctcctc ctgggtacaa gctggttact ggcgacagaa acaattcttc ttgccgcaat 901tacaacaagc aagcaagtga gcaaaactgg gctaattaca gtgcagaaca aaatcgaatg 961gggcaggcgg gaagcaccat ctctaactcc catgcacagc cttttgattt ccccgatgat 1021aaccagaatt ctaaaaaact agctgctgga catgaattac agccactagc cattgtggac 1081cagcgacctt caagcagagc cagcagtcgt gccagcagca gacctcggcc tgatgacctg 1141gagatctag

Therapeutic Agents

Therapeutic agents include pharmaceutically acceptable agents useful inthe treatment of wounds or the promotion of wound-healing, whethercurrently existing and known or later developed. Therapeutic agentsinclude, for example, anti-infectives, anesthetics, analgesics,antibiotics, narcotics, and steroidal and non-steroidalanti-inflammatory agents. Preferred therapeutic agents include topicalsteroid anti-inflammatory agents, antimicrobial agents, local andtopical anesthetics, and topical opioids. In certain embodiments, one,two three, four, five or six therapeutic agents may be used incombination.

Agents Useful for Wound Healing

As used herein, agents useful for wound healing include stimulators,enhancers or positive mediators of the wound healing cascade which 1)promote or accelerate the natural wound healing process or 2) reduceeffects associated with improper or delayed wound healing, which effectsinclude, for example, adverse inflammation, epithelialization,angiogenesis and matrix deposition, and scarring and fibrosis.

Positive mediators, enhancers and stimulators include for example, anagent which may stimulate, enhance, facilitate, or accelerate (i.e.,agonize) the quantity, quality or efficacy of wound healing or theactive wound healing process, or a wound healing-associated growthfactor or cytokine at a wound site, or the activation of a woundhealing-associated growth factor or cytokine receptor. Such agents mayinclude a wound healing-associated growth factor or cytokine or apartially modified form of a wound healing-associated growth factor orcytokine, for example. A partially modified form of woundhealing-associated growth factor or cytokine may, for example, have alonger half-life than the natural wound healing-associated growth factoror cytokine. Alternatively, it may be an inhibitor of woundhealing-associated growth factor or cytokine metabolism.

Partial modification of such an agent may be by way of addition,deletion or substitution of amino acid residues. A substitution may forexample be a conserved substitution. Hence a partially modified moleculemay be a homologue of the molecule from which it was derived. It mayhave at least about 40%, for example about 50, 60, 70, 80, 90 or 95%,homology with the molecule from which it is derived.

As used herein, agents useful for wound healing may include for example,wound-healing-promoting or scar-reducing agents for wound treatmentmodalities now known in the art or later-developed; exemplary factors,agents or modalities including natural or synthetic growth factors,cytokines, or modulators thereof to promote wound healing, wound healingpromoting bioengineered matrix, dressings bandages, and the like.Suitable examples may include, but not limited to 1) topical or dressingand related therapies and debriding agents (such as, for example,Santyl® collagenase) and Iodosorb® (cadexomer iodine); 2) antimicrobialagents, including systemic or topical creams or gels, including, forexample, silver-containing agents such as SAGs (silver antimicrobialgels), (CollaGUARD™, Innocoll, Inc) (purified type-I collagen proteinbased dressing), CollaGUARD Ag (a collagen-based bioactive dressingimpregnated with silver for infected wounds or wounds at risk ofinfection), DermaSIL™ (a collagen-synthetic foam composite dressing fordeep and heavily exuding wounds); 3) cell therapy or bioengineered skin,skin substitutes, and skin equivalents, including, for example,Dermograft (3-dimensional matrix cultivation of human fibroblasts thatsecrete cytokines and growth factors), Apligraf® (human keratinocytesand fibroblasts), Graftskin® (bilayer of epidermal cells and fibroblaststhat is histologically similar to normal skin and produces growthfactors similar to those produced by normal skin), TransCyte (a HumanFibroblast Derived Temporary Skin Substitute) and Oasis® (an activebiomaterial that comprises both growth factors and extracellular matrixcomponents such as collagen, proteoglycans, and glycosaminoglycans); 4)cytokines, growth factors or hormones (both natural and synthetic)introduced to the wound to promote wound healing, including, forexample, NGF, NT3, BDGF, integrins, plasmin, semaphoring, blood-derivedgrowth factor, keratinocyte growth factor, tissue growth factor,TGF-alpha, TGF-beta, PDGF (one or more of the three subtypes may beused: AA, AB, and B), PDGF-BB, TGF-beta 3, factors that modulate therelative levels of TGFβ3, TGFβ1, and TGFβ2 (e.g., Mannose-6-phosphate),sex steroids, including for example, estrogen, estradiol, or anoestrogen receptor agonist selected from the group consisting ofethinyloestradiol, dienoestrol, mestranol, oestradiol, oestriol, aconjugated oestrogen, piperazine oestrone sulphate, stilboestrol,fosfesterol tetrasodium, polyestradiol phosphate, tibolone, aphytoestrogen, 17-beta-estradiol; thymic hormones such asThymosin-beta-4, EGF, HB-EGF, fibroblast growth factors (e.g., FGF1,FGF2, FGF7), keratinocyte growth factor, TNF, interleukins family ofinflammatory response modulators such as, for example, IL-10, IL-1,IL-2, IL-6, IL-8, and IL-10 and modulators thereof; INFs (INF-alpha,-beta, and -delta); stimulators of activin or inhibin, and inhibitors ofinterferon gamma prostaglandin E2 (PGE2) and of mediators of theadenosine 3′,5′-cyclic monophosphate (cAMP) pathway; adenosine A1agonist, adenosine A2 agonist or 5) other agents useful for woundhealing, including, for example, both natural or synthetic homologues,agonist and antagonist of VEGF, VEGFA, IGF; IGF-1, proinflammatorycytokines, GM-CSF, and leptins and 6) IGF-1 and KGF cDNA, autologousplatelet gel, hypochlorous acid (Sterilox® lipoic acid, nitric oxidesynthase3, matrix metalloproteinase 9 (MMP-9), CCT-ETA, alphavbeta6integrin, growth factor-primed fibroblasts and Decorin, silvercontaining wound dressings, Xenaderm™, papain wound debriding agents,lactoferrin, substance P, collagen, and silver-ORC, placental alkalinephosphatase or placental growth factor, modulators of hedgehogsignaling, modulators of cholesterol synthesis pathway, and APC(Activated Protein C), keratinocyte growth factor, TNF, Thromboxane A2,NGF, BMP bone morphogenetic protein, CTGF (connective tissue growthfactor), wound healing chemokines, decorin, modulators of lactateinduced neovascularization, cod liver oil, placental alkalinephosphatase or placental growth factor, and thymosin beta 4. In certainembodiments, one, two three, four, five or six agents useful for woundhealing may be used in combination.

It is to be understood that the agents useful for wound healing(including for example, growth factors and cytokines) above encompassall naturally occurring polymorphs (for example, polymorphs of thegrowth factors or cytokines). Also, functional fragments, chimericproteins comprising one of said agents useful for wound healing or afunctional fragment thereof, homologues obtained by analogoussubstitution of one or more amino acids of the wound healing agent, andspecies homologues are encompassed. It is contemplated that one or moreagents useful for wound healing may be a product of recombinant DNAtechnology, and one or more agents useful for wound healing may be aproduct of transgenic technology. For example, platelet derived growthfactor may be provided in the form of a recombinant PDGF or a genetherapy vector comprising a coding sequence for PDGF.

A fragment or partially modified form thereof refers to a fragment orpartially modified form of the wound healing agent which retains thebiological or wound healing functionality of the factor, although it mayof course have additional functionality. Partial modification may, forexample, be by way of addition, deletion or substitution of amino acidresidues. For example, a substitution may be a conserved substitution.Hence the partially modified molecules may be homologues of the woundhealing agent. They may, for example, have at least about 40% homologywith said factor. They may for example have at least about 50, 60, 70,80, 90 or 95% homology with said factor. For example, in certainembodiments, IL-10 or a fragment or a partially modified form thereofmay be administered at a concentration of between about 1 μM and about10 μM. It may be administered at a concentration of between about 2.5 μMand about 5 μM. In certain other embodiments, IL-10 or a fragment or apartially modified form thereof may be administered immediately prior towound healing, but may be effective if administered within about 7 daysof wounding. It could be administered on at least two occasions.

Gap Junction Modifying Agents

As used herein, “gap junction modifying agent” may broadly include thoseagents or compounds that prevent, decrease or modulate, in whole or inpart, the activity, function, or formation of a hemichannel or a gapjunction.

In other embodiments, a gap junction modifying agent prevents ordecreases, in whole or in part, the formation or activity of ahemichannel or a gap junction.

In certain embodiments, a gap junction modifying agent induces closure,in whole or in part, of a hemichannel or a gap junction. In otherembodiments, a gap junction modifying agent blocks, in whole or in part,a hemichannel or a gap junction. In certain embodiments, a gap junctionmodifying agent decreases or prevents, in whole or in part, the openingof a hemichannel or gap junction.

In certain embodiments, said blocking or closure of a gap junction orhemichannel by a gap junction modifying agent can reduce or inhibitextracellular hemichannel communication by preventing or decreasing theflow of small molecules through an open channel to and from anextracellular or periplamic space.

In certain embodiments, a gap junction modifying agent prevents,decreases or alters the activity or function of a connexin, ahemichannel or a gap junction. As used herein, modification of the gapjunction activity or function may include the opening or closing of gapjunctions, opening or closing of connexon hemichannel, and/or passage ofmolecules or ions through the gap junctions.

In certain another aspect, gap junction modifying agent may include, forexample, aliphatic alcohols; octanol; heptanol; anesthetics (e.g.halothane), ethrane, fluothane, propofol and thiopental; anandamide;arylaminobenzoate (FFA: flufenamic acid and similar derivatives that arelipophilic); carbenoxolone; Chalcone: (2′,5′-dihydroxychalcone); CHFs(Chlorohydroxyfuranones); CMCF(3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone); dexamethasone;doxorubicin (and other anthraquinone derivatives); eicosanoidthromboxane A(2) (TXA(2)) mimetics; NO (nitric oxide); Fatty acids (e.g.arachidonic acid, oleic acid and lipoxygenase metabolites; Fenamates(flufenamic (FFA), niflumic (NFA) and meclofenamic acids (MFA));Genistein; glycyrrhetinic acid (GA):18a-glycyrrhetinic acid and18-beta-glycyrrhetinic acid, and derivatives thereof; lindane;lysophosphatidic acid; mefloquine; menadione;2-Methyl-1,4-naphthoquinone, vitamin K(3); nafenopin; okadaic acid;oleamide; oleic acid; PH, gating by intracellular acidification; e.g.acidifying agents; polyunsaturated fatty acids; fatty acid GJICinhibitors (e.g. oleic and arachidonic acids); quinidine; quinine; alltrans-retinoic acid; and tamoxifen.

Exemplary compounds used for altering gap junction functions (e.g., gapjunction blocking agents, gap junction protein phosphorylating anddephosphorylating agents) have been previously reported by Jensen etal., see U.S. Pat. No. 7,153,822, Larsen et al., see U.S. Pat. No.7,250,397, Gourdie et al., see WO2006069181, and Tudor et al., seeWO2003032964, and other assorted published patent applications.

As used herein, “gap junction phosphorylating agent” or “gap junctiondephosphorylating agent” may include those agents or compounds capableof inducing phosphorylation or de-phosphorylation on connexin residues.Exemplary sites of phosphorylation or dephosphorylation include one ormore of a tyrosine, serine or threonine residue on the connexin protein.In certain embodiments, modulation of phosphorylation may occur on oneor more residues on one or more connexin proteins. Exemplary gapjunction phosphorylating or dephosphorylating agent are well know in theart and may include, for example, c-Src tyrosine kinase or other Gprotein-coupled receptor agonists. See Giepmans B, J. Biol. Chem., Vol.276, Issue 11, 8544-8549, Mar. 16, 2001. In one embodiment, modulationof phosphorylation on one or more of these residues impacts hemichannelfunction, particularly by closing the hemichannel. In anotherembodiment, modulation of phosphorylation on one or more of theseresidues impacts gap junction function, particularly by closing the gapjunction.

Dosage Forms and Formulations and Administration

A therapeutically effective amount of each of the combination partners(e.g. an anti-connexin agent and a wound healing agent) may beadministered simultaneously, separately or sequentially and in anyorder. The agents may be administered separately or as a fixedcombination. When not administered as a fixed combination, preferredmethods include the sequential administration of one or moreanti-connexin agents and one or more agents useful for wound healing,either or both of which are provided in amounts or doses that are lessthat those used when the agent or agents are administered alone, i.e.,when they are not administered in combination, either physically or inthe course of treatment of a wound. Such lesser amounts of agentsadministered are typically from about one-twentieth to about one-tenththe amount or amounts of the agent when administered alone, and may beabout one-eighth the amount, about one-sixth the amount, about one-fifththe amount, about one-fourth the amount, about one-third the amount, andabout one-half the amount when administered alone. Preferably, theagents are administered sequentially within at least about one-half hourof each other. The agents may also be administered with about one hourof each other, with about one day to about one week of each other, or asotherwise deemed appropriate. Preferably, the anti-connexin agent isadministered first. Preferably, where one or more anti-connexin agentsare used, an anti-connexin peptide or anti-connexin peptidomimetic,e.g., an anti-connexin agent that can block or reduce hemichannelopening, is administered prior to the administration of an anti-connexinagent that blocks or reduce connexin expression or the formation ofhemichannels or gap junctions, e.g., by downregulation of connexinprotein expression. Preferably, the anti-connexin agent or agents is/areanti-connexin 43 agent(s).

The agents of the invention of the may be administered to a subject inneed of treatment, such as a subject with any of the diseases orconditions mentioned herein. The condition of the subject can thus beimproved. The anti-connexin agent and combinational partner may thus beused in the treatment of the subject's body by therapy. They may be usedin the manufacture of a medicament to treat any of the conditionsmentioned herein. Thus, in accordance with the invention, there areprovided formulations by which cell-cell communication can bedownregulated in a transient and site-specific manner.

The anti-connexin agent may be present in a substantially isolated form.It will be understood that the product may be mixed with carriers ordiluents which will not interfere with the intended purpose of theproduct and still be regarded as substantially isolated. A product ofthe invention may also be in a substantially purified form, in whichcase it will generally comprise about 90%, e.g. at least about 95%, atleast about 98% or at least about 99% of the polynucleotide (or otheranti-connexin agent) or dry mass of the preparation.

Depending on the intended route of administration, the pharmaceuticalproducts, pharmaceutical compositions, combined preparations andmedicaments of the invention may, for example, take the form ofsolutions, suspensions, instillations, salves, creams, gels, foams,ointments, emulsions, lotions, paints, sustained release formulations,or powders, and typically contain about 0.1%-95% of activeingredient(s), preferably about 0.2%-70%. Other suitable formulationsinclude pluronic gel-based formulations,carboxymethylcellulose(CMC)-based formulations, andhyroxypropylmethylcellulose(HPMC)-based formulations. Other usefulformulations include slow or delayed release preparations.

Gels or jellies may be produced using a suitable gelling agentincluding, but not limited to, gelatin, tragacanth, or a cellulosederivative and may include glycerol as a humectant, emollient, andpreservative. Ointments are semi-solid preparations that consist of theactive ingredient incorporated into a fatty, waxy, or synthetic base.Examples of suitable creams include, but are not limited to,water-in-oil and oil-in-water emulsions. Water-in-oil creams may beformulated by using a suitable emulsifying agent with propertiessimilar, but not limited, to those of the fatty alcohols such as cetylalcohol or cetostearyl alcohol and to emulsifying wax. Oil-in-watercreams may be formulated using an emulsifying agent such as cetomacrogolemulsifying wax. Suitable properties include the ability to modify theviscosity of the emulsion and both physical and chemical stability overa wide range of pH. The water soluble or miscible cream base may containa preservative system and may also be buffered to maintain an acceptablephysiological pH.

Foam preparations may be formulated to be delivered from a pressurizedaerosol canister, via a suitable applicator, using inert propellants.Suitable excipients for the formulation of the foam base include, butare not limited to, propylene glycol, emulsifying wax, cetyl alcohol,and glyceryl stearate. Potential preservatives include methylparaben andpropylparaben.

Preferably the agents of the invention are combined with apharmaceutically acceptable carrier or diluent to produce apharmaceutical composition. Suitable carriers and diluents includeisotonic saline solutions, for example phosphate-buffered saline.Suitable diluents and excipients also include, for example, water,saline, dextrose, glycerol, or the like, and combinations thereof. Inaddition, if desired substances such as wetting or emulsifying agents,stabilizing or ph buffering agents may also be present.

The term “pharmaceutically acceptable carrier” refers to anypharmaceutical carrier that does not itself induce the production ofantibodies harmful to the individual receiving the composition, andwhich can be administered without undue toxicity. Suitable carriers canbe large, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, and amino acid copolymers.

Pharmaceutically acceptable salts can also be present, e.g., mineralacid salts such as hydrochlorides, hydrobromides, phosphates, sulfates,and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like.

Suitable carrier materials include any carrier or vehicle commonly usedas a base for creams, lotions, gels, emulsions, lotions or paints fortopical administration. Examples include emulsifying agents, inertcarriers including hydrocarbon bases, emulsifying bases, non-toxicsolvents or water-soluble bases. Particularly suitable examples includepluronics, HPMC, CMC and other cellulose-based ingredients, lanolin,hard paraffin, liquid paraffin, soft yellow paraffin or soft whiteparaffin, white beeswax, yellow beeswax, cetostearyl alcohol, cetylalcohol, dimethicones, emulsifying waxes, isopropyl myristate,microcrystalline wax, oleyl alcohol and stearyl alcohol.

Preferably, the pharmaceutically acceptable carrier or vehicle is a gel,suitably a nonionic polyoxyethylene-polyoxypropylene copolymer gel, forexample, a Pluronic gel, preferably Pluronic F-127 (BASF Corp.). Thisgel is particularly preferred as it is a liquid at low temperatures butrapidly sets at physiological temperatures, which confines the releaseof the agent to the site of application or immediately adjacent thatsite.

An auxiliary agent such as casein, gelatin, albumin, glue, sodiumalginate, carboxymethylcellulose, methylcellulose, hydroxyethylcelluloseor polyvinyl alcohol may also be included in the formulation of theinvention.

Other suitable formulations include pluronic gel-based formulations,carboxymethylcellulose(CMC)-based formulations, andhyroxypropylmethylcellulose(HPMC)-based formulations. The compositionmay be formulated for any desired form of delivery, including topical,instillation, parenteral, intramuscular, subcutaneous, or transdermaladministration. Other useful formulations include slow or delayedrelease preparations.

Where the anti-connexin agent is a nucleic acid, such as apolynucleotide, uptake of nucleic acids by mammalian cells is enhancedby several known transfection techniques for example those including theuse of transfection agents. Such techniques may be used with certainanti-connexin agents, including polynucleotides. The formulation whichis administered may contain such transfection agents. Examples of theseagents include cationic agents (for example calcium phosphate andDEAE-dextran) and lipofectants (for example Lipofectam™ andTransfectam™), and surfactants.

Where the anti-connexin agent comprises a polynucleotide, conveniently,the formulation further includes a surfactant to assist withpolynucleotide cell penetration or the formulation may contain anysuitable loading agent. Any suitable non-toxic surfactant may beincluded, such as DMSO. Alternatively a transdermal penetration agentsuch as urea may be included.

The effective dose for a given subject or condition can be determined byroutine experimentation or other methods known in the art or laterdeveloped. For example, in order to formulate a range of dosage values,cell culture assays and animal studies can be used. The dosage of suchcompounds preferably lies within the dose that is therapeuticallyeffective for at least 50% of the population, and that exhibits littleor no toxicity at this level.

The effective dosage of each of the anti-connexin agents employed in themethods and compositions of the invention may vary depending on a numberof factors including the particular anti-connexin agent or agentsemployed, the combinational partner, the mode of administration, thefrequency of administration, the condition being treated, the severityof the condition being treated, the route of administration, the needsof a patient sub-population to be treated or the needs of the individualpatient which different needs can be due to age, sex, body weight,relevant medical condition specific to the patient.

The dose at which an anti-connexin agent is administered to a patientwill depend upon a variety of factors such as the age, weight andgeneral condition of the patient, the condition that is being treated,and the particular anti-connexin agent that is being administered.

A suitable dose may be from about 0.001 to about 100 mg/kg body weightsuch as about 0.01 to about 40 mg/kg body weight. A suitable dose mayhowever be from about 0.001 to about 0.1 mg/kg body weight such as about0.01 to about 0.050 mg/kg body weight. Doses from about 1 to 100, 200,300, 400, and 500 micrograms are appropriate.

For example, in certain embodiments, the anti-connexin agent compositionmay be applied at about 0.01 micromolar (μM) or 0.05 μM to about 200 μMfinal concentration at the treatment site and/or adjacent to thetreatment site. Preferably, the antisense polynucleotide composition isapplied at about 0.05 μM to about 100 μM final concentration, morepreferably, the anti-connexin agent composition is applied at about 1.0μM to about 50 μM final concentration, and more preferably, theanti-connexin agent composition is applied at about 5-10 μM to about30-50 μM final concentration. Additionally, the combined anti-connexinagent composition is applied at about 8 μM to about 20 μM finalconcentration, and alternatively the anti-connexin agent composition isapplied at about 10 μM to about 20 μM final concentration, or at about10 to about 15 μM final concentration. In certain other embodiments, theanti-connexin agent is applied at about 10 μM final concentration. Inyet another embodiment, the anti-connexin agent composition is appliedat about 1-15 μM final concentration. Anti-connexin agent dose amountsinclude, for example, about 0.1-1, 1-2, 2-3, 3-4, or 4-5 micrograms(μg), from about 5 to about 10 μg, from about 10 to about 15 μg, fromabout 15 to about 20 μg, from about 20 to about 30 μg, from about 30 toabout 40 μg, from about 40 to about 50 μg, from about 50 to about 75 μg,from about 75 to about 100 μg, from about 100 μg to about 250 μg, andfrom 250 μg to about 500 μg. Dose amounts from 0.5 to about 1.0milligrams or more or also provided, as noted above. Dose volumes willdepend on the size of the site to be treated, and may range, forexample, from about 25-100 μL to about 100-200 μL, from about 200-500 μLto about 500-1000 μL. Milliliter doses are also appropriate for largertreatment sites.

Still other dosage levels between about 1 nanogram (ng)/kg and about 100mg/kg body weight per day of each of the agents described herein. Incertain embodiments, the dosage of each of the subject compounds willgenerally be in the range of about 1 ng to about 1 microgram per kg bodyweight, about 1 ng to about 0.1 microgram per kg body weight, about 1 ngto about 10 ng per kg body weight, about 10 ng to about 0.1 microgramper kg body weight, about 0.1 microgram to about 1 microgram per kg bodyweight, about 20 ng to about 100 ng per kg body weight, about 0.001 mgto about 100 mg per kg body weight, about 0.01 mg to about 10 mg per kgbody weight, or about 0.1 mg to about 1 mg per kg body weight. Incertain embodiments, the dosage of each of the subject compounds willgenerally be in the range of about 0.001 mg to about 0.01 mg per kg bodyweight, about 0.01 mg to about 0.1 mg per kg body weight, about 0.1 mgto about 1 mg per kg body weight, about 1 mg to about 10 mg per kg bodyweight or about 10 mg to about 100 mg per kg body weight. If more thanone anti-connexin agent is used, the dosage of each anti-connexin agentneed not be in the same range as the other. For example, the dosage ofone anti-connexin agent may be between about 0.01 mg to about 10 mg perkg body weight, and the dosage of another anti-connexin agent may bebetween about 0.1 mg to about 1 mg per kg body weight. Other amountswill be known to those of skill in the art and readily determined.

Conveniently, the anti-connexin agent is administered in a sufficientamount to downregulate expression of a connexin protein, or modulate gapjunction formation or connexon opening for at least about 0.5 to 1 hour,at least about 1-2 hours, at least about 2-4 hours, at least about 4-6hours, at least about 6-8 hours, at least about 8-10 hours, at leastabout 12 hours, or at least about 24 hours post-administration.

The dosage of each of the anti-connexin agents in the compositions andmethods of the subject invention may also be determined by reference tothe concentration of the composition relative to the size, length,depth, area or volume of the area to which it will be applied. Forexample, in certain topical and other applications, e.g., instillation,dosing of the pharmaceutical compositions may be calculated based onmass (e.g. micrograms) of or the concentration in a pharmaceuticalcomposition (e.g. μg/μl) per length, depth, area, or volume of the areaof application.

Agents useful for wound healing suitable for the preparation of thepharmaceutical compositions described herein may be prepared andadministered using methods as known in the art (see, for example, U.S.Pat. Nos. 7,098,190, 6,319,907, 6,331,298, 6,387,364, 6,455,569,6,566,339, 6,696,433, 6,855,505, 6,900,181, 7,052,684 and EP1100529 B1.The concentration of each anti-connexin agent and agents useful forwound healing need not be in the same range as the other. Other amountswill be known to those of skill in the art and readily determined. Forexample, suitable combination dosages and formulations in accordancewith various aspects and embodiments as described herein may beadministered according to the dosing regimen as described in U.S. Pat.No. 6,903,078 to Lewis entitled “Combination PDGF, KGF, IGF, and IGFBPfor wound healing.”

The initial and any subsequent dosages administered will depend upon thepatient's age, weight, condition, and the disease, wound, disorder orbiological condition being treated. Depending on the wound healingagent, the dosage and protocol for administration will vary, and thedosage will also depend on the method of administration selected, forexample, local or systemic administration.

The wound healing agent may be applied internally or externally, and maybe directed towards any tissue exhibiting a wound. For topicaladministration of IGF, for example, a zinc oxide formulation can beapplied, which induces the local production of IGF, as described inTarnow et al, Scand J. Plast Reconstr Hand Surg. 28: 255-259 (1994). Aneffective dose of PDGF has been reported to be 5 ng/mm² or higher whenapplied topically as described in U.S. Pat. No. 4,861,757, and at least1 ng/ml local concentration of an isoform of PDGF (for example, PDGF-AA,PDGF-BB, or PDGF-AB), up to about 30 ng/ml local concentration appliedto a population of fibroblasts as described in Lepisto et al., BiochemBiophys Res. Comm 209: 393-399 (1995). PDGF can be administered in acarboxymethylcellulose gel formulation at concentrations of about 10μg/gm to about 500 μg/gm of gel, about 20 μg/gm to about 200 μg/gm, andabout 30 μg/gm to about 100 μg/gm of gel, optimally about 100 μg/gm ofgel. Efficacy of PDGF has been achieved within the range of about 3μg/ml solution to about 300 μg/ml of solution administered.

About 50 μl of KGF of a concentration of about 5 μg/ml may be effectivefor wound healing by topical application to epithelial tissue asdescribed in Sotozono et al, Invest. Opthal. Vis. Science 36: 1524-29(1995). As described in U.S. Pat. No. 4,861,757, an effective amount ofIGF when co-administered with PDGF is in the range of at least 2.5ng/mm² to about 5 ng/mm², with a ratio of PDGF to IGF in the range ofabout 1:10 to about 25:1 weight to weight, with the most effectiveratios being PDGF to IGF of about 1:1 to about 2:1 weight to weight.IGFBP administered in combination with IGF has been shown to increasewound healing at dose levels of about 5 μg of IGF with about 1.5 μg ofphosphorylated IGFBP in a molar ration of about 11:1 IGF:IGFBP, asdescribed in Jyung et al, Surgery 115:233-239 (1994).

For administration of polypeptide therapeutics, for example, PDGF, KGF,IGF and IGFBP polypeptides, the dosage can be in the range of about 5 μgto about 50 μg/kg of tissue to which the application is directed, alsoabout 50 μg to about 5 mg/kg, also about 100 μg to about 500 μg/kg oftissue, and about 200 to about 250 μg/kg. For polynucleotidetherapeutics, for example in a gene therapy administration protocol,depending on the expression strength the polynucleotide in the patient,for tissue targeted administration, vectors containing expressibleconstructs including PDGF, KGF, IGF, and IGFBP coding sequences can beadministered in a range of about 100 ng to about 200 mg of DNA for localadministration in a gene therapy protocol, also about 500 ng to about 50mg, also about 1 μg to about 2 mg of DNA, about 5 μg of DNA to about 500μg of DNA, and about 20 μg to about 100 μg during a local administrationin a gene therapy protocol, and about 250 μg, per injection oradministration. Factors such as method of action and efficacy oftransformation and expression are therefore considerations that willeffect the dosage required for ultimate efficacy for administration ofDNA therapeutics. Where greater expression is desired, over a largerarea of tissue, larger amounts of DNA or the same amountsre-administered in a successive protocol of administrations, or severaladministrations to different adjacent or close tissue portions of forexample, a wound site may be required to effect a positive therapeuticoutcome.

Therapeutic agents and gap junction modifying agents suitable for thepreparation of the pharmaceutical compositions described herein may beformulated and administered using methods as known in the art. Theinitial and any subsequent dosages administered will depend upon thepatient's age, weight, condition, and the disease, wound, disorder orbiological condition being treated. Depending on the therapeutic, thedosage and protocol for administration will vary, and the dosage willalso depend on the method of administration selected, for example, localor systemic administration.

As noted herein, the doses of either an anti-connexin agent or anotheragent administered in combination can be adjusted down from the dosesadministered when given alone.

The combined use of several agents may reduce the required dosage forany individual agent because the onset and duration of effect of thedifferent agents may be complementary. In a preferred embodiment, thecombined use of one or more anti-connexin agents and one or moretherapeutic agents, agents useful for wound healing, and/or gap junctionmodifying agents has an additive, synergistic or super-additive effect.

In some cases, the combination of one or more anti-connexin agents andone or more therapeutic agents and/or one or more agents useful forwound healing, and/or one or more gap junction modifying agents have anadditive effect. In other cases, the combination can havegreater-than-additive effect. Such an effect is referred to herein as a“supra-additive” effect, and may be due to synergistic or potentiatedinteraction.

The term “supra-additive promotion of wound healing” refers to a meanwound healing produced by administration of a combination of ananti-connexin agent and one or more therapeutic agents, agents usefulfor wound healing and/or gap junction modifying agents, is statisticallysignificantly higher than the sum of the wound healing produced by theindividual administration of either any of the agents alone. Whetherproduced by combination administration of an anti-connexin agent and oneor more therapeutic agents, agents useful for wound healing and/or gapjunction modifying agents is “statistically significantly higher” thanthe expected additive value of the individual compounds may bedetermined by a variety of statistical methods as described hereinand/or known by one of ordinary skill in the art. The term “synergistic”refers to a type of supra-additive inhibition in which both theanti-connexin agent and one or more therapeutic agents, agents usefulfor wound healing and/or gap junction modifying agents individually havethe ability to promote wound healing or reduce fibrosis and scarring.The term “potentiated” refers to type of supra-additive effect in whichone of the anti-connexin agent or one or more therapeutic agents, agentsuseful for wound healing and/or gap junction modifying agentsindividually has the increased ability to promote wound healing.

In general, potentiation may be assessed by determining whether thecombination treatment produces a mean wound healing increase in atreatment group that is statistically significantly supra-additive whencompared to the sum of the mean wound healing increases produced by theindividual treatments in their treatment groups respectively. The meanwound healing increase may be calculated as the difference betweencontrol group and treatment group mean wound healing. The fractionalincrease in wound healing, “fraction affected” (Fa), may be calculatedby dividing the treatment group mean wound healing increase by controlgroup mean wound healing. Testing for statistically significantpotentiation requires the calculation of Fa for each treatment group.The expected additive Fa for a combination treatment may be taken to bethe sum of mean Fas from groups receiving either element of thecombination. The Two-Tailed One-Sample T-Test, for example, may be usedto evaluate how likely it is that the result obtained by the experimentis due to chance alone, as measured by the p-value. A p-value of lessthan 0.05 is considered statistically significant, that is, not likelyto be due to chance alone. Thus, Fa for the combination treatment groupmust be statistically significantly higher than the expected additive Fafor the single element treatment groups to deem the combination asresulting in a potentiated supra-additive effect.

Whether a synergistic effect results from a combination treatment may beevaluated by the median-effect/combination-index isobologram method(Chou, T., and Talalay, P. (1984) Ad. Enzyme Reg. 22:27-55). In thismethod, combination index (CI) values are calculated for differentdose-effect levels based on parameters derived from median-effect plotsof the anti-connexin agent alone, the one or more agents useful forwound healing alone, and the combination of the two at fixed molarratios. CI values of & It; 1 indicate synergy, CI-1 indicates anadditive effect, and CP1 indicates an antagonistic effect. This analysismay be performed using computer software tools, such as CalcuSyn,Windows Software for Dose Effect Analysis (Biosoft(D, Cambridge UK).

Any method known or later developed in the art for analyzing whether asupra-additive effect exists for a combination therapy is contemplatedfor use in screening for suitable anti-connexin agents for use incombination with one or more therapeutic agents, agents useful for woundhealing and/or gap junction modifying agents.

In another preferred embodiment, the combined use of one or moreanti-connexin agents and one or more therapeutic agents, agents usefulfor wound healing, and/or gap junction modifying agents reduces theeffective dose of any such agent compared to the effective dose whensaid agent administered alone. In certain embodiments, the effectivedose of the agent when used in combination with one or moreanti-connexin agents is about 1/15 to about ½, about 1/10 to about ⅓,about ⅛ to about ⅙, about ⅕, about ¼, about ⅓ or about ½ the dose of theagent when used alone.

In another preferred embodiment, the combined use of one or moreanti-connexin agents and one or more therapeutic agents, agents usefulfor wound healing, and/or gap junction modifying agents reduces thefrequency in which said agent is administered compared to the frequencywhen said agent is administered alone. Thus, these combinations allowthe use of lower and/or fewer doses of each agent than previouslyrequired to achieve desired therapeutic goals.

The doses may be administered in single or divided applications. Thedoses may be administered once, or application may be repeated.

One or more anti-connexin agents and one or more therapeutic agents,agents useful for wound healing and/or gap junction modifying agents maybe administered by the same or different routes. The various agents ofthe invention can be administered separately at different times duringthe course of therapy, or concurrently in divided or single combinationforms.

In one aspect of the invention the anti-connexin agent is administeredin one composition and the therapeutic agent, wound healing agent and/orgap junction modifying agent is administered in a second composition. Inone embodiment the first composition comprising one or moreanti-connexin agents is administered before the second compositioncomprising one or more therapeutic agents, agents useful for woundhealing and/or gap junction modifying agents. In one embodiment thefirst composition comprising one or more anti-connexin agents isadministered after the second composition comprising one or moretherapeutic agents, agents useful for wound healing and/or gap junctionmodifying agents. In one embodiment the first composition comprising oneor more anti-connexin agents is administered before and after the secondcomposition comprising one or more therapeutic agents, agents useful forwound healing and/or gap junction modifying agents. In one embodimentthe first composition comprising one or more anti-connexin agents isadministered about the same time as the second composition comprisingone or more therapeutic agents, agents useful for wound healing and/orgap junction modifying agents.

Preferably one or more anti-connexin agents and one or more therapeuticagents, agents useful for wound healing and/or gap junction modifyingagents are delivered by topical administration (peripherally or directlyto a site), including but not limited to topical administration usingsolid supports (such as dressings and other matrices) and medicinalformulations (such as gels, mixtures, suspensions and ointments). In oneembodiment, the solid support comprises a biocompatible membrane orinsertion into a treatment site. In another embodiment, the solidsupport comprises a dressing or matrix. In one embodiment of theinvention, the solid support composition may be a slow release solidsupport composition, in which the one or more anti-connexin agents andone or more therapeutic agents, agents useful for wound healing and/orgap junction modifying agents is dispersed in a slow release solidmatrix such as a matrix of alginate, collagen, or a syntheticbioabsorbable polymer. Preferably, the solid support composition issterile or low bio-burden. In one embodiment, a wash solution comprisingone or more anti-connexin agents can be used.

The delivery of one or more anti-connexin agents and one or moretherapeutic agents, agents useful for wound healing and/or gap junctionmodifying agents of the formulation over a period of time, in someinstances for about 1-2 hours, about 2-4 hours, about 4-6 hours, about6-8, or about 24 hours or longer, may be a particular advantage in moresevere injuries or conditions. In some instances, cell loss may extendwell beyond the site of a procedure to surrounding cells. Such loss mayoccur within 24 hours of the original procedure and is mediated by gapjunction cell-cell communication. Administration of anti-connexinagent(s), e.g., for downregulation of connexin expression, or blockadeof connexon opening, therefore will modulate communication between thecells, or loss into the extracellular space in the case of connexonregulation, and minimize additional cell loss or injury or consequencesof injury.

While the delivery period will be dependent upon both the site at whichthe downregulation is to be induced and the therapeutic effect which isdesired, continuous or slow-release delivery for about 1-2 hours, about2-4 hours, about 4-6 hours, about 6-8, or about 24 hours or longer isprovided. In accordance with the present invention, this is achieved byinclusion of the anti-connexin agents and/or one or more therapeuticagents, agents useful for wound healing and/or gap junction modifyingagents in a formulation together with a pharmaceutically acceptablecarrier or vehicle, particularly in the form of a formulation forcontinuous or slow-release administration.

As noted, the one or more agents of the invention may be administeredbefore, during, immediately following wounding, for example, or withinabout 180, about 120, about 90, about 60, or about 30 days, butpreferably within about 10, about 9, about 8, about 7, about 6, about 5,about 4, about 3, or about 2 days or less, and most preferably withinabout 24, about 12, about 10, about 9, about 8, about 7, about 6, about5, about 4, about 3, about 2 hours or within about 60, about 45, about30, about 15, about 10, about 5, about 4, about 3, about 2, about 1minute following wounding, for example.

The routes of administration and dosages described herein are intendedonly as a guide since a skilled physician will determine the optimumroute of administration and dosage for any particular patient andcondition.

Any of the methods of treating a subject having or suspected of havingor predisposed to, or at risk for, a disease, disorder, and/orcondition, referenced or described herein may utilize the administrationof any of the doses, dosage forms, formulations, and/or compositionsherein described.

Dressings and Matrices

In one aspect, the one or more anti-connexin agents, one or moretherapeutic agents, agents useful for wound healing and/or gap junctionmodifying agents are provided in the form of a dressing or matrix. Incertain embodiments, the one or more agents of the invention areprovided in the form of a liquid, semi solid or solid composition forapplication directly, or the composition is applied to the surface of,or incorporated into, a solid contacting layer such as a dressing gauzeor matrix. The dressing composition may be provided for example, in theform of a fluid or a gel. The one or more anti-connexin agents and oneor more therapeutic agents, agents useful for wound healing and/or gapjunction modifying agents may be provided in combination withconventional pharmaceutical excipients for topical application. Suitablecarriers include: Pluronic gels, Polaxamer gels, Hydrogels containingcellulose derivatives, including hydroxyethyl cellulose, hydroxymethylcellulose, carboxymethyl cellulose, hydroxypropylmethyl cellulose andmixtures thereof; and hydrogels containing polyacrylic acid (Carbopols).Suitable carriers also include creams/ointments used for topicalpharmaceutical preparations, e.g., creams based on cetomacrogolemulsifying ointment. The above carriers may include alginate (as athickener or stimulant), preservatives such as benzyl alcohol, buffersto control pH such as disodium hydrogen phosphate/sodium dihydrogenphosphate, agents to adjust osmolarity such as sodium chloride, andstabilizers such as EDTA.

Suitable dressings or matrices may include, for example, the followingwith one or more anti-connexin agents with one or more therapeuticagents, agents useful for wound healing and/or gap junction modifyingagents:

1) Absorptives: suitable absorptives may include, for example,absorptive dressings, which can provide, for example, a semi-adherentquality or a non-adherent layer, combined with highly absorptive layersof fibers, such as for example, cellulose, cotton or rayon.Alternatively, absorptives may be used as a primary or secondarydressing.

2) Alginates: suitable alginates include, for example, dressings thatare non-woven, non-adhesive pads and ribbons composed of naturalpolysaccharide fibers or xerogel derived from seaweed. Suitablealginates dressings may, for example, form a moist gel through a processof ion exchange upon contact with exudate. In certain embodiments,alginate dressings are designed to be soft and conformable, easy topack, tuck or apply over irregular-shaped areas. In certain embodiments,alginate dressings may be used with a second dressing.

3) Antimicrobial Dressings: suitable antimicrobial dressings mayinclude, for example, dressings that can facilitate delivery ofbioactive agents, such as, for example, silver and polyhexamethylenebiguanide (PHMB), to maintain efficacy against infection, where this isneeded or desirable. In certain embodiments, suitable antimicrobialdressings may be available as for example, as sponges, impregnated wovengauzes, film dressings, absorptive products, island dressings, nylonfabric, non-adherent barriers, or a combination of materials.

4) Biological & Biosynthetics: suitable biological dressings orbiosynthetic dressings may include, for example, gels, solutions orsemi-permeable sheets derived from a natural source. In certainembodiments, a gel or solution is applied to the treatment site andcovered with a dressing for barrier protection. In another embodiment, asheet is placed in situ which may act as membrane, remaining in placeafter a single application.

5) Collagens: suitable collagen dressings may include, for example,gels, pads, particles, pastes, powders, sheets or solutions derived fromfor example, bovine, porcine or avian sources or other natural sourcesor donors. In certain embodiments, the collagen dressing may interactwith treatment site exudate to form a gel. In certain embodiments,collagen dressing may be used in combination with a secondary dressing.

6) Composites: suitable composite dressings may include, for example,dressings that combine physically distinct components into a singleproduct to provide multiple functions, such as, for example, a bacterialbarrier, absorption and adhesion. In certain embodiment, the compositedressings are comprised of, for example, multiple layers and incorporatea semi- or non-adherent pad. In certain embodiment, the composite mayalso include for example, an adhesive border of non-woven fabric tape ortransparent film. In certain other embodiment, the composite dressingmay function as for example, either a primary or a secondary dressingand in yet another embodiment, the dressing may be used in combinationwith topical pharmaceutical composition.

7) Contact Layers: suitable contact layer dressings may include, forexample, thin, non-adherent sheets placed on an area to protect tissuefrom for example, direct contact with other agents or dressings appliedto the treatment site. In certain embodiments, contact layers may bedeployed to conform to the shape of the area of the treatment site andare porous to allow exudate to pass through for absorption by anoverlying, secondary dressing. In yet another embodiment, the contactlayer dressing may be used in combination with topical pharmaceuticalcomposition.

8) Elastic Bandages: suitable elastic bandages may include, for example,dressings that stretch and conform to the body contours. In certainembodiment, the fabric composition may include for example, cotton,polyester, rayon or nylon. In certain other embodiments, the elasticbandage may for example, provide absorption as a second layer ordressing, to hold a cover in place, to apply pressure or to cushion atreatment site.

9) Foams: suitable foam dressings may include, for example, sheets andother shapes of foamed polymer solutions (including polyurethane) withsmall, open cells capable of holding fluids. Exemplary foams may be forexample, impregnated or layered in combination with other materials. Incertain embodiment, the absorption capability may be adjusted based onthe thickness and composition of the foam. In certain other embodiments,the area in contact with the treatment site may be non-adhesive for easyremoval. In yet another embodiment, the foam may be used in combinationwith an adhesive border and/or a transparent film coating that can serveas an anti-infective barrier.

10) Gauzes & Non-Woven dressings: suitable gauze dressings and wovendressings may include, for example, dry woven or non-woven sponges andwraps with varying degrees of absorbency. Exemplary fabric compositionmay include, for example, cotton, polyester or rayon. In certainembodiment, gauzes and non-woven dressing may be available sterile ornon-sterile in bulk and with or without an adhesive border. Exemplarygauze dressings and woven dressings may be used for cleansing, packingand covering a variety of treatment sites.

11) Hydrocolloids: suitable hydrocolloid dressings may include, forexample, wafers, powders or pastes composed of gelatin, pectin orcarboxymethylcellulose. In certain embodiment, wafers are self-adheringand available with or without an adhesive border and in a wide varietyof shapes and sizes. Exemplary hydrocolloids are useful on areas thatrequire contouring. In certain embodiments, powders and pasteshydrocolloids may use used in combination with a secondary dressing.

12) Hydrogels (Amorphous): suitable amorphous hydrogel dressings mayinclude, for example, formulations of water, polymers and otheringredients with no shape, designed to donate moisture and to maintain amoist healing environments and or to rehydrate the treatment site. Incertain embodiment, hydrogels may be used in combination with asecondary dressing cover.

13) Hydrogels: Impregnated Dressings: suitable impregnated hydrogeldressings may include, for example, gauzes and non-woven sponges, ropesand strips saturated with an amorphous hydrogel. Amorphous hydrogels mayinclude for example, formulations of water, polymers and otheringredients with no shape, designed to donate moisture to a drytreatment site and to maintain a moist healing environment.

14) Hydrogel Sheets: suitable hydrogel sheets may include for example,three-dimensional networks of cross-linked hydrophilic polymers that areinsoluble in water and interact with aqueous solutions by swelling.Exemplary hydrogels are highly conformable and permeable and can absorbvarying amounts of drainage, depending on their composition. In certainembodiment, the hydrogel is non-adhesive against the treatment site ortreated for easy removal.

15) Impregnated Dressings: suitable impregnated dressings may include,for example, gauzes and non-woven sponges, ropes and strips saturatedwith a solution, an emulsion, oil, gel or some other pharmaceuticallyactive compound or carrier agent, including for example, saline, oil,zinc salts, petrolatum, xeroform and scarlet red as well as thecompounds described herein.

16) Silicone Gel Sheets: suitable silicone gel sheet dressings mayinclude, for example, soft covers composed of cross-linked polymersreinforced with or bonded to mesh or fabric.

17) Solutions: suitable liquid dressings may include, for example,mixtures of multiprotein material and other elements found in theextracellular matrix. In certain embodiment, exemplary solutions may beapplied to the treatment site after debridement and cleansing and thencovered with an absorbent dressing or a nonadherent pad.

18) Transparent Films: suitable transparent film dressings may includepolymer membranes of varying thickness coated on one side with anadhesive. In certain embodiments, transparent films are impermeable toliquid, water and bacteria but permeable to moisture vapor andatmospheric gases. In certain embodiments, the transparency allowsvisualization of the treatment site.

19) Fillers: suitable filler dressings may include, for example, beads,creams, foams, gels, ointments, pads, pastes, pillows, powders, strandsor other formulations. In certain embodiment, fillers are non-adherentand may include a time-released antimicrobial. Exemplary fillers may beuseful to maintain a moist environment, manage exudate, and fortreatment of for example, partial- and full-thickness wounds, infectedwounds, draining wounds and deep wounds that require packing.

Combination Wound Treatment

General Aspects

The present invention is directed to pharmaceutical compositions andtheir methods of use wherein the composition comprises therapeuticallyeffective amounts of one or more anti-connexin agents and one or moretherapeutic agents, agents useful for wound healing and/or gap junctionmodifying agents. The compositions are useful in enhancing or promotinghealing of wounds, such wounds include wounds which may be slow to healor refractory to conventional wound treatment or wound healing promotingtherapies.

Equally, in instances of other tissue damage (particularly wounds) themethods and compositions of the invention are effective in promoting thewound healing process, reducing swelling and inflammation, and inminimizing scar formation. The formulations have clear benefit in thetreatment of wounds, whether the result of external trauma (includingburns), internal trauma, or surgical intervention, as well as chronicwounds.

Compositions

Accordingly, in one aspect, the invention provides compositions for usein therapeutic treatment, which comprises: at least one anti-connexinagent and at least one therapeutic agent, and/or wound healing agentand/or gap junction modifying agent. In a preferred embodiment, thecomposition further comprises a pharmaceutically acceptable carrier orvehicle.

In one preferred form, the composition contains one or more antisensepolynucleotides to the mRNA of one connexin protein only. In anotherpreferred form, the composition comprises one or more anti-connexinpeptides or pepidomimetics. Most preferably, this connexin protein isconnexin 43.

The compositions may comprise polynucleotides or anti-connexin peptidesto more than one connexin protein. Preferably, one of the connexinproteins to which polynucleotides or anti-connexin peptides are directedis connexin 43. Other connexins to which the polynucleotides oranti-connexin peptides are directed may include, for example, connexins26, 30, 30.3, 31.1, 32, 36, 37, 40, 40.1, 44.6, 45 and 46. Suitableexemplary polynucleotides (and ODNs) directed to various connexins areset forth in Table 1. Suitable anti-connexin peptides are also providedherein.

Exemplary Combinations

Exemplary combinations of an anti-connexin agent and a wound healingagent according to the compositions and methods of the present inventioninclude the following:

(a) one of the following anti-connexin agents:

-   -   a connexin 43 antisense polynucleotide;    -   a connexin 43 antisense ODN;    -   a polynucleotide comprising SEQ. ID. NO: 2;    -   an ODN comprising SEQ. ID. NO: 2;    -   a polynucleotide comprising SEQ. ID. NO: 1;    -   an ODN comprising SEQ. ID. NO: 1:    -   a connexin 43 RNAi polynucleotide; or    -   a connexin 43 siRNA polynucleotide; and

(b) one of the following agents useful for wound healing:

-   -   Activin;    -   FGF-2 or fibroblast growth factor 2;    -   FGF-1 or fibroblast growth factor-1;    -   VEGF or vascular endothelial growth factor;    -   GM-SF or granulocyte monocyte stimulating factor;    -   Platelet factor 4;    -   EGF or epidermal growth factor;    -   TGF or transforming growth factor beta (e.g. TGF-1,2,3);    -   TNF alpha or tumor necrosis factor alpha;    -   IL-1 interleukin-1;    -   IL-4 interleukin-4;    -   IL-7 interleukin-7;    -   IL-8 interleukin-8;    -   IL-10 interleukin-10;    -   GMCSF or granulocyte-macrophage/colony-stimulating factor;    -   CTGF or Connective tissue growth factor;    -   Thymosin beta 4;    -   IGF-1 or insulin-like growth factor-1;    -   PDGF or platelet-derived growth factor;    -   PDGF-BB or platelet-derived growth factor BB;    -   Mannose-6-phosphate;    -   (aFGF) Acidic fibroblast growth factor    -   (bFGF) Basic fibroblast growth factor    -   (HB-EGF) Heparin binding epidermal growth factor    -   (hGH) Human growth hormone    -   (KGF) Keratinocyte growth factor    -   (KGF-2) Keratinocyte growth factor-2    -   (MMP) Matrix metalloproteinase    -   (PDECGF) Platelet-derived endothelial cell growth factor    -   (PDEGF) Platelet-derived epidermal growth factor    -   (rbbFGF) Recombinant bovine basic fibroblast growth factor    -   (rhbFGF) Recombinant human basic fibroblast growth factor    -   (rhPDGF-BB) Recombinant human platelet-derived growth factor        (BB-dimer)    -   (TGF-alpha) Transforming growth factor-alpha    -   (TGF-beta) Transforming growth factor-beta    -   (TIMP) Tissue inhibitor of matrix metalloproteinase    -   (VEGF) Vascular endothelial growth factor    -   (IGFBP) Insulin-Like Growth Factor Binding Protein (e.g.,        IGFBP-2, IGFBP-3, IGFBP-4); and,    -   (FGF) or fibroblast growth factors (e.g. FGF-1, FGF-2).

This listing of combinations is exemplary in nature. Other combinationsof anti-connexin agents and therapeutic agents described herein are alsoincluded. Other combinations of anti-connexin agents and agents usefulfor wound healing described herein are also included. Other combinationsof anti-connexin agents and gap junction modifying agents describedherein are also included.

Kits, Medicaments and Articles of Manufacturer

Optionally, one or more anti-connexin agents and one or more agentsuseful for wound healing, therapeutic agents, and/or gap junctionmodifying agents (e.g., peptides, proteolytic inhibitors, extracellularmatrix components, fragments and peptides, steroids, cytokines, oxygendonators or vitamins) may also be used in the manufacture of themedicament.

In one aspect, the invention provides a kit comprising one or morecompositions or formulations described. For example, the kit may includea composition comprising an effective amount of one or moreanti-connexin agents and one or more therapeutic agents, agents usefulfor wound healing and/or gap junction modifying agents.

Articles of manufacturer are also provided, comprising a vesselcontaining a composition or formulation of the invention as describedherein and instructions for use for the treatment of a subject. Forexample, In another aspect, the invention includes an article ofmanufacture comprising a vessel containing a therapeutically effectiveamount of one or more pharmaceutically acceptable anti-connexin agentsand one or more pharmaceutically acceptable therapeutic agents forpromotion or improvement of wound healing and instructions for use,including use for the treatment of a subject.

Treatment

The compositions and formulations of the invention may be used inconjunction or combination with a composition for promoting the healingof wounds or to reduce swelling, inflammation and/or scarring. Thecompositions and formulations of the invention may also be used inconjunction or combination with a composition for promoting and/orimproving the healing of acute or chronic wounds. In one aspect, thewound will be the result of surgery or trauma.

Suitable therapeutic agents, agents useful for wound healing, and/or gapjunction modifying agents used in combination with one or moreanti-connexin agents for the treatment of wounds are described herein.In one aspect, the anti-connexin agent may be administered incombination with an agent useful for wound healing such as a chemokine,a cytokine, growth factor, or combination thereof. In one embodiment,the chemokine is a chemokine ligand 2 (Ccl2) antagonist. In anotherembodiment, the cytokine is a tumor necrosis factor alpha (TNF-α)antagonist. In another embodiment, the cytokine is IL-10. In oneembodiment, the growth factor is TGF-beta-3. According to anotherembodiment the wound healing agent is thymosin β4. In yet anotherembodiment, the anti-connexin agent may be administered in combinationwith one or more of IGFBP-2, IGF-7, IGF-1 or modulators thereof, toincrease hydroxyproline and collagen type 1α1 synthesis for thepromotion of wound healing.

Suitable anti-connexin agents for use in combination with an agentuseful for wound healing, a therapeutic agent or a gap-junctionmodifying agent are described herein and may include, for example, apolynucleotide such as an ODN, a blocker or other connexin binding agentsuch as, for example, receptor mimetic peptide; an absorber to removeactivity, such as, for example, synthetically expressed receptormolecules or mimetics; or antibodies; as wells as, for examples, otheragents useful to adjust gap junction closing/opening, such as compoundsthat phosphorylate or dephosphorylate gap junctions.

In one aspect the invention is directed to a method of promoting orimproving wound healing in a subject, comprising administration atherapeutically effective amount of one or more anti-connexin agents andone or more therapeutic agents, agents useful for wound healing and/or agap junction modifying agents or compounds useful for promoting orimproving healing. In certain embodiments, the administration of one ormore anti-connexin agents and one or more therapeutic agents, agentsuseful for wound healing and/or a gap junction modifying agents, iseffective to reduce inflammation, reduce neutrophil and/or macrophageinfiltration into the wound, reduce granulation tissue deposition,promote cell migration to accelerate wound closure and healing, tofacilitate epitheliam growth, tissue engineering, and surface recoveryfor burns, or any combination thereof. In certain embodiments, theadministration of one or more anti-connexin agents and one or moretherapeutic agents, agents useful for wound healing and/or a gapjunction modifying agents, is effective to reduce or prevent scarformation. In methods to promote or improve scar formation, theanti-connexin agent is preferably administered in combination with, orafter or prior to, administration of TGF-beta-3 or IL-10 ormannose-6-phosphate.

In one aspect the invention is directed to a method of promoting orimproving wound healing in a subject, comprising administration of oneor more anti-connexin agents and one or more therapeutic agents, agentsuseful for wound healing and/or a gap junction modifying agents, in anamount effective to regulate epithelial basal cell division and growth.In one embodiment, the anti-connexin agent is a connexin antisensepolynucleotide effective to regulate epithelial basal cell division andgrowth. In one embodiment, the connexin antisense polynucleotide is aconnexin 26 antisense polynucleotide, a connexin 43 antisensepolynucleotide, or a mixture thereof.

In one aspect the invention is directed to a method of promoting orimproving wound healing, comprising administration of one or moreanti-connexin agents and one or more therapeutic agents, agents usefulfor wound healing and/or a gap junction modifying agents, in an amounteffective to regulate outer layer keratin secretion. In one embodiment,the anti-connexin agent is a connexin antisense polynucleotide effectiveto regulate outer layer keratin secretion. In one embodiment, theconnexin antisense polynucleotide is a connexin 43 antisensepolynucleotide, a 31.1 antisense polynucleotide, or a mixture thereof.

In yet a further aspect, the invention provides a method of decreasingscar formation and/or improving scar appearance in a patient who hassuffered a wound, e.g., a surgical wound (such as in, for example,cosmetic and other surgeries), which comprises the step of administeringone or more anti-connexin agents and one or more therapeutic agents,agents useful for wound healing and/or a gap junction modifying agentsto said wound to downregulate expression of one or more connexinprotein(s) at and immediately adjacent the site of said wound. Again,the wound may be the result of trauma or surgery, with the formulationbeing applied to the wound immediately prior to surgical repair and/orclosure thereof. As noted herein, in methods to reduce or improve scarformation or appearance, the anti-connexin agent is preferablyadministered in combination with, or after or prior to, administrationof a suitable amount of TGF-beta-3 or IL-10 or mannose-6-phosphate.

In one aspect the invention is directed to a method of reducing,preventing or ameliorating tissue damage in a subject, comprisingadministration of one or more anti-connexin agents and one or moretherapeutic agents, agents useful for wound healing and/or a gapjunction modifying agents.

In a further aspect, the invention is directed to a method of reducingswelling and/or inflammation as part of treating a wound and/or tissuesubjected to physical trauma which comprises the step of administeringan anti-connexin composition or formulation as defined above and one ormore therapeutic agents, agents useful for wound healing, and/or gapjunction modifying agents to or proximate to said wound or tissue. Inone embodiment the wound is the result of physical trauma to tissue,including neuronal tissue such as the brain, spinal cord or optic nerve,or skin or eye.

In one aspect the invention is directed to sustained administration ofone or more anti-connexin agents and one or more therapeutic agents,agents useful for wound healing, and/or gap junction modifying agents.In one embodiment, an anti-connexin agent is administered for at leastabout 1-24 hours, at least about 2, hours, at least about 3 hours, atleast about 4 hours, at least about 5 hours, at least about 6 hours, atleast about 7 hours, at least about 8 hours, at least about 9 hours, atleast about 10 hours, at least about 11 hours, at least about 12 hoursor at least about 24 hours. In one embodiment, connexin expression isdownregulated over a sustained period of time. Preferably connexin 43expression is downregulated for a sustained period of time.Conveniently, connexin 43 expression is downregulated for at least about2, 4, 6, 8, 10, 12, or 24 hours. According to one embodiment, the woundis a chronic wound. Suitable subjects include a diabetic subject.

In one aspect, the present invention provides a method of treating asubject having a wound which comprises sustained administration of aneffective amount of an anti-connexin agent and one or more therapeuticagents, agents useful for wound healing, and/or gap junction modifyingagents to the wound. In a further aspect, the present invention providesa method of promoting or improving wound healing in a subject whichcomprises sustained administration of one or more anti-connexin agentsand one or more therapeutic agents, agents useful for wound healing,and/or gap junction modifying agents to a wound. In a further aspect,the present invention provides a method of reducing, preventing orameliorating swelling and/or inflammation in a subject which comprisessustained administration of one or more anti-connexin agents and one ormore therapeutic agents, agents useful for wound healing, and/or gapjunction modifying agents to a wound. In a further aspect, the presentinvention provides a method of reducing, preventing or ameliorating scarformation in a subject which comprises sustained administration of oneor more anti-connexin agents and one or more therapeutic agents, agentsuseful for wound healing, and/or gap junction modifying agents to awound.

According to another further aspect, the present invention provides amethod of promoting or improving wound healing in a subject having awound which comprises sustained administration of an anti-connexincomposition or formulation of the present invention and one or moretherapeutic agents, agents useful for wound healing, and/or gap junctionmodifying agents to a wound area in an amount effective to increasere-epithlialization rates in the wound area. In one embodiment themethod comprises sustained administration of a connexin 43 antisensepolynucleotide and/or a connexin 31.1 antisense polynucleotide and oneor more therapeutic agents, wound healing agents and/or gap junctionmodifying agents. In one embodiment, the composition or compositions areadministered in a sustained release formulation. In another embodiment,the composition or compositions are administered for a sustained periodof time. Conveniently, the composition is effective to decrease connexin43 and/or 31.1 levels or activity (e.g., hemichannel or gap junctionactivity) for at least about 24 hours. According to one embodiment, thewound is a chronic wound. Subjects which may be treated include diabeticsubjects.

In yet another aspect, the present invention provides invention providesa method of promoting or improving wound healing in a subject having awound which comprises sustained administration of an anti-connexincomposition or formulation of the present invention and one or moretherapeutic agents, agents useful for wound healing, and/or gap junctionmodifying agents to a wound area in an amount effective to effective toregulate epithelial basal cell division and growth and/or effective toregulate outer layer keratin secretion. In one embodiment, thecomposition comprises a connexin antisense polynucleotide effective toregulate epithelial basal cell division or growth, preferably a connexin26 antisense polynucleotide, a connexin 43 antisense polynucleotide, ora mixture thereof. In one embodiment, the composition comprises aconnexin antisense polynucleotide effective to regulate outer layerkeratinization, preferably, a connexin 31.1 antisense polynucleotide. Inone embodiment, the composition or compositions are administered in asustained release formulation. In another embodiment, the composition orcompositions are administered for a sustained period of time.Conveniently, the composition is effective to decrease connexin 43, 26,and/or 31.1 levels for at least about 24 hours. According to oneembodiment, the wound is a chronic wound. Subjects which may be treatedinclude diabetic subjects.

In another aspect, methods for treating a subject having a chronic woundare provided. Such methods include administering to the subject ananti-connexin agent capable of inhibiting the expression, formation, oractivity of a connexin, or a connexin hemichannel, in combination withone or more therapeutic agents, wound healing agents and/or gap junctionmodifying agents.

In one aspect the invention is directed to a method for treatment orprophylaxis of a chronic wound comprising administering to a subject inneed thereof an effective amount of an anti-connexin agent administeredto said chronic wound or a tissue associated with said chronic wound incombination with one or more therapeutic agents, agents useful for woundhealing and/or gap junction modifying agents. In another embodiment, thechronic wound is a chronic skin wound and a composition of the presentinvention is administered to the skin or a tissue associated with theskin of said subject for an effective period of time. A chronic skinwound suitable for treatment may, for example, be selected from thegroup consisting of pressure ulcers, diabetic ulcers, venous ulcers,arterial ulcers, vasculitic ulcers, and mixed ulcers. The chronic woundmay be an arterial ulcer which comprises ulcerations resulting fromcomplete or partial arterial blockage. The chronic wound may be a venousstasis ulcer which comprises ulcerations resulting from a malfunction ofthe venous valve and the associated vascular disease. The chronic woundmay be a trauma-induced ulcer.

In one embodiment, the anti-connexin agent is administered incombination with a growth factor. Preferably the growth factor is PDGF,EGF, or FGF (e.g., FGF-2).

When not administered as a fixed combination, preferred methods includethe sequential administration of one or more anti-connexin agents andone or more growth factors. Preferably, the agents are administeredsequentially within at least about one-half hour of each other. Theagents may also be administered with about one hour of each other, withabout one day to about one week of each other, or as otherwise deemedappropriate. Preferably, the anti-connexin agent is administered first.Preferably, where one or more anti-connexin agents are used, ananti-connexin peptide or anti-connexin peptidomimetic, e.g., ananti-connexin agent that can block or reduce hemichannel opening, isadministered prior to the administration of an anti-connexin agent thatblocks or reduce connexin expression or the formation of hemichannels orgap junctions, e.g., by downregulation of connexin protein expression.Preferably, the anti-connexin agent or agents is/are anti-connexin 43agent(s).

In another embodiment for treatment of wounds, including chronic wounds,either or both of the one or more anti-connexin agents and one or moregrowth factors are provided in amounts or doses that are less that thoseused when the agent or agents are administered alone, i.e., when theyare not administered in combination, either physically or in the courseof treatment of a wound. Such lesser amounts of agents administered aretypically from about one-twentieth to about one-tenth the amount oramounts of the agent when administered alone, and may be aboutone-eighth the amount, about one-sixth the amount, about one-fifth theamount, about one-fourth the amount, about one-third the amount, andabout one-half the amount when administered alone.

In one embodiment the method for treatment or prophylaxis of a chronicwound comprises sustained administration of one or more anti-connexinagents and one or more therapeutic agents, agents useful for woundhealing and/or gap junction modifying agents. In one embodiment, thecomposition or compositions are administered in a sustained releaseformulation. In another embodiment, the composition or compositions areadministered for a sustained period of time. Conveniently, thecomposition is effective to decrease connexin 43 levels, or block orreduce connexin 43 hemichannel opening, for at least about 1-2 hours,about 2-4 hours, about 4-6 hours, about 4-8 hours, about 12 hours, about18 hours, or about 24 hours. Subjects which may be treated includediabetic subjects, and patients with other ulcers, including venousulcers and others described herein and known in the art.

The following examples which will be understood to be provided by way ofillustration only and not to constitute a limitation on the scope of theinvention.

EXAMPLES Example 1

Wound healing was assessed using a model with 1.6 mm diameter fullthickness excisional wounds on 8 week old mice. Followed byimmunohistochemistry and histology, with RT-PCR analysis of geneexpression.

Skin wounds repair by a combination of re-epithelializing action and,connective tissue contraction followed by an angiogenic response whichleads to a dense network of blood vessels in the wound granulationtissue (Grose, R. and Werner, S. (2004). Wound-healing studies intransgenic and knockout mice. Mol Biotechnol 28, 147-66.). A robustinflammatory response commences soon after any tissue damage. This bothprotects the wound from microbial infection and produces many kinds ofbioactive substances that act on the host cells at the wound site. Avariety of inflammatory cells migrate into the wound fulfilling severaldifferent functions. Neutrophils are the earliest leukocytes to berecruited to the wound and their main role is to defend the host frominvasion by microbes, which they do by releasing toxic free oxygenradicals and secreting proinflammatory cytokines. Subsequently,macrophages clear away spent neutrophils and other cell andextracellular matrix debris at the wound site. Macrophages are also themajor producers of cytokines, chemokines and growth factors that willdirect subsequent cell and tissue migration of the repair response.Whilst many of the signals regulating the inflammation and tissue repairprocess are clearly diffusible and operate over long distances, localcell-cell communication via cell adhesion molecules and cell-celljunctions appears also to play a significant role.

One junctional link between cells which may play a significantregulatory role is the gap junction which is a hexameric channel formedof proteins from the connexin family. Gap junctions are reported to beexpressed by almost all cells in the body (Wei, C. J., Xu, X. and Lo, C.W. (2004). Annu Rev Cell Dev Biol 20, 811-38.) and have been reported tomediate changes in cell migration.

The level of connexin 43 (Cx43) protein at the epidermal wound edge hasbeen reported to naturally decreases over 24-48 hours. DownregulatingCx43 protein levels by application of antisense oligodeoxynucleotides(asODN) to skin wound and burn injury sites has been reported to lead tosignificantly accelerated healing compared with control senseoligodeoxynucleotides (sODN) treated wounds (Qiu, C., Coutinho, P.,Frank, S., Franke, S., Law, L. Y., Martin, P., Green, C. R. and Becker,D. L. (2003). Targeting connexin 43 expression accelerates the rate ofwound repair. Curr Biol 13, 1697-703; Coutinho, P., Qiu, C., Frank, S.,Wang, C. M., Brown, T., Green, C. R. and Becker, D. L. (2005). Limitingburn extension by transient inhibition of connexin 43 expression at thesite of injury. Br J Plast Surg 58, 658-67).

The experiments showed that acute downregulation of Cx43 protein at awound site led to an increase in keratinocyte proliferation andmigration, and in the rate at which fibroblasts migrate into the woundand lay down collagen matrix. We noted a decrease in neutrophilinfiltration and a concomitant reduction in chemokine ligand 2 (Ccl2)and cytokine tumor necrosis factor alpha (TNF-α) mRNA. Subsequently, wealso noted a reduced recruitment of macrophages which may be aconsequence of damping down of the initial inflammatory response, whichis known to have downstream effects on the ensuing wound healingprocess. Together these modified responses resulted in significantlyimproved wound healing.

Wound Model and ODN Treatment

Male, 8 week old, ICR mice were used in the following experiments. Allthe mice were kept under specific pathogen-free conditions in anenvironmentally controlled clean room at University College London andall the experiments were carried out under UK Home Office regulations.Mice were anaesthetized by halothane inhalation. Four full-thicknessexcisional wounds of 6 mm diameter were made on the shaved back oneither side of the dorsal midline with a 6 mm biopsy punch (KaiIndustries). To each pair of wounds a single topical application of 50μl of 1 μM Cx43 as ODN (SEQ. ID No. 2) (Sigma-Genosys) in 30% PluronicF-127 gel (Sigma-Aldrich), chilled on ice, was made to one wound, and anidentical application of 1 μM sense control sODNs was made to the other.Cx43 asODNs application results in a significant knockdown of Cx43protein at the site of delivery within two hours (Becker, D. L.,McGonnell, I., Makarenkova, H. P., Patel, K., Tickle, C., Lorimer, J.and Green, C. R. (1999). Roles for alpha 1 connexin in morphogenesis ofchick embryos revealed using a novel antisense approach. Dev Genet 24,33-42; McGonnell, I. M., Green, C. R., Tickle, C. and Becker, D. L.(2001). Connexin 43 gap junction protein plays an essential role inmorphogenesis of the embryonic chick face. Dev Dyn 222, 420-38; Qiu, C.,Coutinho, P., Frank, S., Franke, S., Law, L. Y., Martin, P., Green, C.R. and Becker, D. L. (2003). Targeting connexin 43 expressionaccelerates the rate of wound repair. Curr Biol 13, 1697-703). Eachwound region was digitally photographed at the indicated time intervals,and the areas of the wounds were calculated. All wound areas wereexpressed as percentages of the initial wound areas. In some series ofexperiments, wounds and their surrounding area, including the scab andepithelial margins, were harvested with an 8 mm biopsy punch (KaiIndustries) at the indicated time intervals after mice were killed withan overdose of chloroform. A minimum of eight mice were used for eachtime point examined.

Histology and Immunostaining

Wound tissues were fixed in 4% formaldehyde buffered with PBS, andembedded in paraffin. Sections (6 μm thick) were subjected tohematoxylin and eosin staining or immunostaining. Measurement ofgranulation tissue area in H&E was performed using Improvision Openlab™4.0.2 software (Improvision). For immunohistochemistry, deparaffinizedsections were treated with endogenous peroxidase blocking reagent (DakoCytomation A/S) and proteinase K (Dako Cytomation A/S) for 20 minutesand 6 minutes at room temperature, respectively. They were thenincubated with rabbit antimyeloperoxidase (MPO) polyclonal antibody(NeoMarkers) diluted 1:200, rat anti-mouse F4/80 monoclonal antibody(mAb) (Abcom Limited) diluted 1:400 or rat antimouse CD31 (plateletendothelial cell adhesion molecule 1, PECAM-1) mAb (PharMingen) orrabbit anti-mouse TGF-β1 polyclonal antibody (Santa Cruz Biotechnology,Inc) both diluted 1:200 overnight at 4° C. after blocking with 15%skimmed milk for 1 hour at room temperature. In addition, some sectionswere reacted with phalloidin-tetramethylrhodamine B isothiocyanate(Sigma-Aldrich) diluted 1:500 for 1 hour at room temperature. Theantibodies were appropriately diluted in Antibody Diluent withBackground Reducing Components (Dako Cytomation A/S). The sectionsreacted with anti-MPO antibody and anti TGF-β1 antibody were stainedwith EnVision+™ (Dako Cytomation A/S) to enhance the signal, accordingto the manufacturer's instructions. The sections that had been reactedwith anti-F4/80 and anti-CD31 antibodies were incubated withbiotinylated rabbit anti-rat immunoglobulin (Dako Cytomation A/S)diluted 1:200 for 1 hour at 37° C. The signal was then enhanced usingthe Catalyzed Signal Amplification System® (Dako Cytomation A/S)according to the manufacturer's instructions. Thereafter,counterstaining was performed with methyl green (Dako Cytomation A/S)followed by MPO, TGF-β31, F4/80, and CD31 staining or4′,6-diamidino-2-phenylindole (DAPI) followed by phalloidin staining.

Immunostaining for connexin 43, blood vessels or α smooth muscle actinwas carried out on cryostat sections of wounds. Sections were fixed inacetone at 4° C. for 5 minutes prior to blocking for 45 minutes.Incubation in primary antibody was for one hour at the followingdilutions: rabbit anti-Cx43 (Sigma) 1:3,000; isoLectin B_FITC 1:2000;von Willebrand Factor (rabbit Dako) 1:400; anti-α smooth muscle actin(Sigma) 1:400 at room temperature. Sections were washed for 3×5 minutesin PBS before a one hour incubation in anti-rabbit-FITC secondaryantibody (Dako) 1:200 at room temperature. Washing 3×5 minutes in PBS,in some cases with 1 μM bis-benzimide (Sigma) in the first wash as anuclear counter stain, and mounted in Citifluor (Citifluor, London, UK).Sections were imaged by confocal microscopy with all parameters keptconstant to allow direct comparison of digital images.

TUNEL Staining

Wound tissues were fixed in 4% formaldehyde buffered with PBS, andembedded in paraffin and sectioned. Deparaffinized sections were treatedwith proteinase K (Dako Cytomation A/S) for 5 minutes at roomtemperature. They were then stained using the In Situ Cell DeathDetection Kit (Roche) according to the manufacture's instructions.Thereafter, counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI). TUNEL stained section were imaged andpositive cells were counted in the granulation tissue in three randomfields, in the two sides and center of each wound (each field was 0.332mm2).

Detection of Proliferating Cells by Labeling with5′-bromo-2′-deoxy-uridine (BrdU)

In another experiment, wounded mice were injected intraperitoneally with1 ml of BrdU (Sigma) in PBS solution (1 mg/ml) 2 hours before harvestingon days 1, 2, and 7. Wound tissues were fixed in 4% formaldehydebuffered with PBS, and embedded in paraffin. Deparaffinized sections (6μm thick) were treated with a HistoMouse™-Plus Kit (ZYMED Laboratories,Inc) to reduce background signals according to manufacturer'sinstructions. Sections were stained with BrdU Detection Kit (BDBioscience Pharmingen) according to manufacture's instruction.Thereafter, counterstaining was performed with methyl green (DakoCytomation A/S).

Measurement of Neutrophils, Macrophages, Fibroblasts, BrdU-PositiveCells, and Angiogenesis

A treatment-blinded observer counted MPO-positive neutrophils andF4/80-positive macrophages in the wound bed (defined as the areasurrounded by unwounded skin, fascia, regenerated epidermis, and eschar)in 3 random high-power fields of 0.332 mm². BrdU-positive cells in thewound margin and the nascent epidermis regions of eachimmunohistochemically stained section were counted as describedpreviously and expressed per 100 m of epidermis (Mori, R., Kondo, T.,Nishie, T., Ohshima, T. and Asano, M. (2004). Impairment of skin woundhealing in beta-1,4-galactosyltransferase-deficient mice with reducedleukocyte recruitment. Am J Pathol 164, 1303-14). Number of fibroblastlike cells (phalloidin-positive cells with spindle-shape body) at thewound margin were counted in high-power fields also (each field was0.332 mm²). The neovascularization was followed using von Wildebrandfactor fluorescent staining of endothelial cells at days 5, 7, 10 and 14after wounding. Myofibroblasts were identified by anti-α smooth muscleactin staining. For quantification of both fluorescent stains a confocalmicroscope was used to take single section images from comparable zonesfrom a minimum six animals per time point. All parameters of imageacquisition were kept constant to allow comparison. Images were madebinary at a standard threshold and positive pixels were counted usingImage J (NIH Image).

Hydroxyproline Analysis

The collagen content of the wound area was assessed by determining theamount of hydroxyproline (HP), a major component of collagen. Sampleswere homogenized in 1 ml of T-PER® Tissue Protein Extraction Reagent(PIERCE Biotechnology Inc.) including Halt™ Protease Inhibitor Cocktail,EDTA-Free (PIERCE Biotechnology Inc.), and were centrifuged at 15,000rpm for 20 minutes at 4° C. to remove the debris. Concentrations ofprotein were measured using a BCA™ Protein Assay Kit (PIERCEBiotechnology Inc.), and the amounts of HP were determined with Sircol™Soluble Collagen Assay Kit (Biocolor Ltd.). The data were expressed asamounts of HP/total protein (ng/g) for each sample.

Cell Culture

Swiss 3T3 fibroblasts were grown in Dulbecco's modified Eagle's medium(DMEM; Gibco) supplemented with 10% fetal calf serum (Labtech) and 1%penicillinstreptomycin solution (Sigma, Poole) in a 5% CO2 incubator at37° C. Unless otherwise stated, the cells were maintained in this mediumfor most experiments. The cells were passaged by trypsinization and usedat passages 6-10 at a confluency of ≈90%. Cells were plated on 13 mmglass coverslips in 24-well dishes (Nunc) with 4-5×10⁵ cells per wellcontaining 1 ml of medium. Cells reached confluency after 72 hours andwere then used for experimentation.

Cell Migration Assay by Wounding

The fibroblast cell assay involved creating a “wound” in a confluentmonolayer of fibroblast cells. This widely used in vitro techniquemimics the behaviour of migrating cells in vivo (Lampugnani, M. G.(1999) Cell Migration into a wounded area in vitro. Methods Mol Biol 96:177-182). Wounding was performed by drawing a microelectrode across thecoverslip, producing a lesion of standard width. Phase contrast imageswere acquired using a ×5 objective on a Zeiss inverted microscope withan incubation chamber at 370 and 5% CO2. An image of a defined area atthe edge of a cover slip was taken immediately after wounding and afurther image was taken of the same area 4 hours later, a time at whichmigration could be clearly seen to have taken place (FIG. 7E, 7F). Aminimum of eight coverslips, were imaged in each of the control andtreated groups. Migration was quantified by image analysis, measuringthe change in wound area (pixels) using Image J software (NIH).

In order to knock down Cx43 expression in the fibroblasts the media wasreplaced with serum-free DMEM containing either 20 μM asODNs or 20 μMcontrol sODNs. This was incubated for two hours before being replacedwith serum containing DMEM. The wounding assay was then carried out asabove. Previous experience has shown that two hours is sufficient toachieve significant knockdown of Cx43 protein (Qiu, C., Coutinho, P.,Frank, S., Franke, S., Law, L. Y., Martin, P., Green, C. R. and Becker,D. L. (2003). Targeting connexin 43 expression accelerates the rate ofwound repair. Curr Biol 13, 1697-703) and the knockdown processcontinues during the wound migration assay.

Isolation of RNA and Quantitative Gene Expression Level with Real-TimePCR

Total RNA was extracted from skin wound samples using TRIzol Reagent(Invitrogen), according to the manufacturer's instructions. Tenmicrograms of total RNA was reverse-transcribed into cDNA using theSuperScript First-Strand Synthesis System for RT-PCR (Invitrogen).

Gene specific primers and probe were obtained as TaqMan® Gene ExpressionAssays (Applied Biosystems) for Cx43, Ccl2, Colα1, TNFα and TGFβ1. Theenzyme and buffer system was purchased as TaqMan® Universal PCR MasterMix (Applied Biosystems). Each sample was analyzed in duplicate.Amplification and real-time detection was performed in the DNA EngineOpticon®2 (MJ Research Inc.). Expression of target genes was comparedwith GAPDH expression.

Statistical Analysis

Statistical differences were determined using the unpaired Student's ttest or the Mann Whitney U test as appropriate. All data are presentedas the mean±s.e.m. Criterion levels for the individual tests are givenin Results.

Downregulation of Cx43 at Wound Sites with Cx43 asODN

Cx43 was found to be predominantly expressed in the lower and middlespinous cell layers of the epidermis and in fibroblasts, blood vesselsand dermal appendages of intact skin. Six hours after the injury Cx43was expressed in hyperproliferative epidermis but began to bedownregulated in the leading edge keratinocytes. Delivery of Cx43 asODNfrom the time of injury markedly reduced protein levels of Cx43 in theepidermis and dermis within two hours of treatment. Such a rapidknockdown is possible because Cx43 protein is turned over rapidly,sometimes within 1.5-2 hours. In order to quantify the extent of Cx43protein and mRNA knockdown and recovery after asODN treatment moreprecisely, we compared expression levels of Cx43 mRNA at treated versusuntreated wound sites with real-time PCR (RT-PCR; FIG. 1). One day afterinjury, expression of Cx43 mRNA at Cx43 asODN treated wounds wassignificantly reduced by comparison with control sODN-treated wounds(2.95 versus 4.7 units, respectively, a 37% reduction; P<0.05). By 7days after the injury, however, expression levels were similar in thetwo wound regimes (4.6 versus 5.2 units for asODN and controlsODN-treated, respectively). Immunostaining of wounds for Cx43 at 1 day,2 days and 7 days after wounding revealed very low levels of Cx43protein in the epidermis and dermis of the Cx43 asODN-treated wound edgeat day 1 compared to controls (FIG. 1). By day 2, some Cx43 staining hadreturned to the dermis of the Cx43 asODN-treated wound but the level wasstill very low in the epidermis. By day 7, in agreement with the RT-PCRfindings, there was no obvious difference in Cx43 staining betweentreated and untreated. These results confirm that this Cx43 asODN, whendelivered by Pluronic gel, does indeed inhibit expression of Cx43 mRNAat early time points after wounding.

Accelerated Closure and Increased Proliferation in Cx43 asODN TreatedWounds

Wounds were identically photographed macroscopically and their areasmeasured digitally. As we have reported previously (Qiu, C., Coutinho,P., Frank, S., Franke, S., Law, L. Y., Martin, P., Green, C. R. andBecker, D. L. (2003). Targeting connexin 43 expression accelerates therate of wound repair. Curr Biol 13, 1697-703), Cx43 asODN-treated woundswere significantly smaller, drier, less inflamed and closed faster thancontrol wounds at days 1 and 2. By day 7, scabs covered the wounds andmade it impossible to give accurate measurements of wound closure.Reepithelialization from the wound edge commenced soon after injury inorder to cover the denuded site. Cx43 asODN treatment of both excisionaland incisional wounds results in wounds that re-epithelialise morerapidly than control ODN treated wounds. We therefore examined whetherthis might be partially due to enhanced proliferation of keratinocytesand fibroblasts in the healing skin of Cx43 asODN treated wounds (FIGS.2A to 2H). Whilst there was little difference in keratinocyteproliferation between control and treated groups in the epidermal woundmargin (FIG. 2E), we showed that, indeed, there are significantlyincreased numbers of BrdU-positive cells in the nascent epidermis ofCx43 asODN-treated wounds after both 2 days and 7 days (FIG. 2F).Similarly, counts of BrdU-positive cells in the dermal wound marginrevealed slightly more cells following asODN-treatment and significantlymore in the granulation tissue at days 1 and 2 (FIGS. 2G and 2H). Theseresults are consistent with our gross microscopic observations, andsuggest that acute downregulation of Cx43 at wound sites leads to asurge of proliferation of wound-edge keratinocytes that continues asthey re-epithelialize the wound. This enhanced proliferation maycontribute to the accelerated re-epithelialization and enhancedgranulation tissue maturation in asODN treated wounds.

Reduced Influx of Inflammatory Cells in Cx43 asODN-TreatedWounds—Neutrophils and Macrophages

Several leukocyte lineages infiltrate the wound site with varying timecourses during the inflammatory response to tissue damage. The twoprimary cell lineages are neutrophils and macrophages both of these canexert profound effects on various aspects of the repair process. We havepreviously evaluated neutrophil influx in Cx43 asODN-treated wounds andhere we confirm with an anti-MPO antibody that their numbers aresignificantly reduced on day 1 and 2, at a stage when neutrophil numbersare peaking in control treated wounds (FIG. 3). There is now clearevidence that the macrophage influx at a wound site may be linked to therate of re-epithelialization and to the eventual extent of scarring atthe wound site, so we have investigated macrophage numbers using F4/80immunohistochemistry (FIGS. 4A to 4C). We found that the number ofmacrophages at Cx43 asODN treated wound sites was significantly reducedat 2 and 7 days after the injury compared with control sODN-treatedwounds, this being a reduction of 33% on day 2 and 32% on day 7 (FIG.4C). These data clearly indicate that acute knockdown of Cx43 at thetime of wounding leads to a dramatic subsequent reduction in both theearly neutrophil and later, macrophage, and inflammatory phases.

Reduced Expression of Ccl2 and TNF-α in Cx43 asODN-Treated Wounds

Neutrophils and macrophages at the wound site release a large variety ofproinflammatory cytokines and chemokines that act directly on cells inthat site (keratinocytes, fibroblasts and endothelial cells) and amplifythe wound inflammatory response. To examine how the reduced influx ofinflammatory cells, after Cx43 knockdown, influences the level of thesesignals, we analysed Ccl2 and TNF-α as a representative chemokine andcytokine, respectively. To quantify expression levels of Ccl2 and TNF-αwe performed RT-PCR analysis on wound tissue on days 1, 2, and 7 (FIGS.5A and 5B). Both mRNAs were robustly upregulated in control sODN-treatedwound sites on day 1, and both peaked in expression levels at day 2,after which their levels decreased. By comparison, expression levels ofCcl2 and TNF-α in Cx43 asODN treated wounds were significantly reduced(P<0.05) on day 2 (Ccl2) and 7 (TNF-α). These results indicate thatreduced recruitment of neutrophils and macrophages in Cx43 asODN treatedwounds was indeed accompanied by diminished expression of thesesignaling molecules without compensation by other cell types.

To quantify expression levels of Ccl2 and TNF-α RT-PCR analysis wasperformed on wound tissue on days 1, 2, and 7. Both mRNAs were robustlyupregulated in control sODN-treated wound sites on day 1, and bothpeaked in expression levels at day 2, after which their levelsdecreased. By comparison, expression levels of Ccl2 and TNF-α in Cx43asODN treated wounds were significantly reduced (P<0.05) on day 2 (Ccl2)and 7 (TNF-α). These results indicate that reduced recruitment ofneutrophils and macrophages in Cx43 asODN treated wounds was indeedaccompanied by diminished expression of these signalling moleculeswithout compensation by other cell types.

Increased TGF-β1 Expression at Cx43 asODN-Treated Wound Sites

The wound-associated growth factor, TGF-β1 has been reported to play awide variety of roles at many stages of the wound-healing process.Therefore, we analyzed the expression levels of TGF-β1 at control sODNand Cx43 asODN treated wound sites with RT-PCR at 1 day, 2 days and 7days after wounding (FIG. 6). TGF-β1 was at low levels on days 1 and 7with no difference between control and treated wounds. However, on day 2after the injury, the expression of TGF-β1 in asODN-treated wounds wassignificantly increased (P<0.05) compared with control sODN-treatedwounds. Immunostaining for TGF-β1 at 2 days revealed TGF-β1 positivecells in the dermis both at the wound site and in the adjacent tissues.Most of the cells were round and had the appearance of leucocytes.However, in the Cx43 asODN-treated tissue an additional TGF-β1 positivecell type could be seen in large numbers at the dermal margins of thewound. These cells appeared to be elongated and more fibroblast like intheir morphology. Interestingly, in the epidermis of Cx43 asODN treatedwounds TGF-β1 appeared to stain much more strongly than in control woundepidermis (FIG. 6.) These results raise the possibility that increasedexpression of TGF-β1 might contribute to some of the changes we see inwound healing following Cx43 asODN treatment.

Granulation Tissue Formation and Maturation

Connective-tissue wound contraction has been said to be a key componentof the skin repair process. This step is closely associated withmigration of fibroblasts into the wound bed and their differentiationinto contractile myofibroblasts followed by their loss (Martin, P.(1997). Science 276, 75-81). Using rhodamine phalloidin combined withDAPI nuclear counter stain we found a significant increase (means of39.4 in control and 99.2 in asODN; P<0.01) in the number of elongatedfibroblast like cells at the margin of 2 day Cx43 asODN-treated, bycomparison to control wounds (FIGS. 7A and B). These data suggest thatthe influx of fibroblasts to form wound granulation tissue is enhancedin wounds when Cx43 protein has been reduced. This may be due to bothenhanced migration and the significantly greater cell proliferation thatwe see in the granulation tissue of asODN-treated wounds.

To investigate whether the enhanced rate of fibroblast migration was dueto a reduction in Cx43 protein expression we used a fibroblastwound-healing assay. Here we knocked down Cx43 protein by applying Cx43asODNs to confluent cultures of fibroblasts two hours prior to a scrapewound assay. Because Cx43 protein is rapidly turned over (with a halflife as short as 1.5-2 hours) this is sufficient to produce a 95%knockdown of the protein within two hours, which can last between eightand 48 hours depending on the cell type. Fibroblast cultures that weretreated with the Cx43 asODNs exhibited a significantly (P=0.02) enhancedrate of wound closure, compared to controls (FIG. 7D) which is entirelyconsistent with our in vivo findings. This strongly suggests thatknockdown of Cx43 protein enhances the rate of migration of fibroblastsboth in vitro and in vivo, and thereby promotes the rate of granulationtissue formation.

When measuring granulation tissue areas, we found that treated tissuewas slightly smaller than untreated tissue at day 5, but this differencewas not significant. However, we found that on days 7, 10 and 14 afterwounding Cx43 asODN treated wounds exhibit significantly (*P<0.05;**P<0.01) smaller areas of granulation tissue than control sODN treatedwounds.

When measuring granulation tissue areas, we found that treated tissuewas slightly smaller than untreated tissue at day 5, but this differencewas not significant. However, we found that on days 7, 10 and 14 afterwounding Cx43 asODN treated wounds exhibit significantly (*P<0.05;**P<0.01) smaller areas of granulation tissue than control sODN treatedwounds (FIG. 9). To investigate how the more rapid contraction of thegranulation tissue was brought about we stained sections with a TUNELlabeling kit, to look for apoptotic cell death, or with anti-smoothmuscle actin (SMA) as a marker of myofibroblasts. Although there werealways slightly fewer apoptotic cells in treated granulation tissue, wefound no significant differences in the numbers of TUNEL positive cellsbetween control and treated animals at 5, 7 and 10 days after wounding(FIG. 10). However, we observed a highly significant difference in theexpression of SMA staining between the two groups at all of these timepoints (FIG. 11). At day 5, staining for SMA could be detected at theedges of the granulation tissue of Cx43 asODN-treated wounds but nostaining could be seen as yet in control wounds. By day 7 staining forSMA could be detected at the edges of the granulation tissue of controlwounds and throughout the granulation tissue of Cx43 asODN-treatedwounds (FIGS. 11 A and 11B). Quantification of the staining at the edgesof the granulation tissue revealed that it was significantly higher(P=0.004) in Cx43 asODN-treated wounds (FIG. 11 E). At 10 days afterwounding most of the staining for SMA had gone from the edges of thegranulation tissue of Cx43 asODN treated wounds, just a little remainedin the center of the wound. This was significantly different(P=0.000002) from control wounds, which showed strong SMA expressionthroughout the granulation tissue (FIGS. 11 C and 11D). These findingsimply that differentiation of fibroblasts into myofibroblasts occursearlier in Cx43 asODN treated wounds and that these cells go on tocontract the wound and are lost much faster than in control wounds. Itwould appear that Cx43 asODN-treated wounds are 2-3 days more advancedthan controls in the maturation of their granulation tissue.

Angiogenesis

Besides the influx of fibroblasts, the other major cellular componentsof wound granulation tissue are the endothelial cells of new bloodvessels. We therefore performed immunohistochemical staining usinganti-CD31 and von Willebrand factor antibodies or isolectinB-FITC inorder to evaluate angiogenesis at treated wound sites at 5, 7, 10 and 14days after wounding (FIGS. 12A to 12H). At 5 days, no blood vesselstaining could be seen in the granulation tissue of control woundswhereas staining was seen in the edges of the granulation tissue of allCx43 asODN treated wounds. At day 7, fine blood vessels were foundthroughout the entire granulation tissue in five out of six Cx43asODN-treated wounds but they had only just started to enter the edgesof the granulation tissue of controls. However, whilst the blood vesselsof asODN-treated wounds were more pervasive they appeared to besignificantly smaller or thinner than those of controls at this timepoint (FIG. 12A). This meant that, when blood vessel staining wasquantified at 7 and 10 days there was significantly more staining incontrols (7 days P=0.0019; 10 days P=0.015) where the vessels werebigger. At 14 days, the size of blood vessels and extent of stainingwere very similar in both treated and control groups (FIG. 12E, 12F).These findings suggest that angiogenesis takes place much earlier afterCx43 asODN treatment. Taken with our other findings relating togranulation tissue maturation it would seem that the treatment enhancesthe rate of wound maturation by 2-3 days.

As shown in the results, exemplary pathological and biologicalconsequences of acute downregulation of Cx43 at sites of skin woundhealing using Cx43 asODN delivered from a Pluronic F-127 slow-releasegel are presented herein. The treatment rapidly down-regulates Cx43protein in the woundsite epidermis and dermis for at least 24 hours withsome return of dermal expression by 48 hours and no obvious differencesbetween groups after 7 days. We show that Cx43 asODN treatment leads tomarkedly accelerated skin wound healing, coincident with reducedleukocyte infiltration, reduced cytokines, increasedre-epithelialization and enhanced wound contraction.

Inflammation

The initial response to wounding is typically the formation of a bloodclot, which, together with local damaged tissue, releasesproinflammatory signals, which trigger inflammatory cell infiltration inthe form of neutrophils and then macrophages into the wound site. Thesesignals and those from the invading inflammatory cells influence bothre-epithelialisation and connective tissue contraction of the wound(Martin, P. (1997). Science 276, 75-81). The migration and infiltrationof inflammatory cells into the wound is associated with cell-cell andcell-matrix interactions and with vasodilation of blood vessels in theproximity of the wound. It has recently been reported that Cx43 isexpressed in activated leukocytes, and at leukocyte-leukocyte andleukocyte-endothelial cell contact sites during their extravasationunder inflammatory conditions, and that functional Cx43 channels areinvolved in release of cytokines and immunoglobulins (reviewed by:Oviedo-Orta, E. and Evans, W. H. (2004). Biochim Biophys Acta 1662,102-12). The results reported here show that numbers of both neutrophilsand macrophages were significantly reduced in Cx43 asODN-treated woundswhich is in keeping with the requirement for Cx43 expression forneutrophil extravasation and release of proinflammatory cytokines. Inaddition, the results show that the chemokine Ccl2 and cytokine TNF-α,which are reported to be chemoattractants for neutrophils andmonocytes/macrophages (Rossi, D. and Zlotnik, A. (2000). Annu RevImmunol 18, 217-42), are also both reduced after Cx43 asODN treatment onday 2 and 7, respectively. Clearly, the reduced levels of these andother growth factors, chemokines, and cytokines at the wound site areindicative of that these agents useful for wound healing are importantmediators (both upstream and downstream) of the reduced influx ofneutrophils and other inflammatory cells.

Several recent reports have postulated that a normal inflammatoryresponse is not essential for skin wound healing (Martin, P. andLeibovich, S. J. (2005). Trends Cell Biol.). PU.1 null mice, which aregenetically missing neutrophils and macrophages, have been said torepair skin lesions without scarring and at a similar rate to, or fasterthan, their wild type siblings (Martin, P., D'Souza, D., Martin, J.,Grose, R., Cooper, L., Maki, R. and McKercher, S. R. (2003). Curr Biol13, 1122-8). Similarly, decreasing the numbers of neutrophils at woundsites by applying antineutrophil sera has been reported to lead tofaster re-epithelialization (Dovi, J. V., He, L. K. and DiPietro, L. A.(2003). J Leukoc Biol 73, 448-55). Correlation between the increase inthe epidermal migration and proliferative capacity and the reducedinflammatory response and the possible role of Cx43 knockdown in theleading-edge keratinocytes and fibroblasts. Furthermore, Cx43 knockdownmay lead to reduced leukocyte influx. The target tissue for Cx43 asODNaction may include endothelial cells or leukocytes, both of which havebeen reported to require Cx43 expression for effective extravasation anda robust inflammatory response (Oviedo-Orta, E., Hoy, T. and Evans, W.H. (2000). Immunology 99, 578-90; Oviedo-Orta, E., Gasque, P. and Evans,W. H. (2001). FASEB J 15, 768-74). Based on these results, alteration ofCx43 protein expression in endothelial cells or leukocytes may alsoregulate the inflammatory response.

TGF-β1

In Cx43 asODN treated wounds we found that mRNA for TGF-β1 issignificantly increased on day 2 compared with controls but is found atrelatively low levels in both treated and control wounds on day 1 andday 7. Expression of TGF-11 has been reported as being associated withmany key events in the wound healing process. Its reported activitiesinclude being an immunosuppressive, promoting fibroblast migration andproliferation, enhancing wound contraction, enhancing granulation tissueformation, enhancing collagen synthesis and deposition, stimulatingangiogenesis and promoting re-epithelialization. The effect of TGF-β1 onthe wound has been reported to be somewhat dependent on dosage andwounding model. Various results have also been reported based onperturbation of different parts of the TGF-β1 signaling pathway andexperiments using genetically modified. For example, in TGF-β1 knockoutmice that are deficient in T cells and B cells (Scid^(−/−) mice) woundhealing is delayed (Crowe, M. J., Doetschman, T. and Greenhalgh, D. G.(2000) J. Invest. Dermatol 115:3-11). However, when the TGF-β1 signalingpathway is disrupted, as it is in Smad3 knockout mice, wound healing isaccelerated (Ashcroft, G. S., Yang, X, Glick, A. B., Weinstein, M.,Letterio, J. L., Mizel, D. E., Anzano, M., Greenwell-Wild, T., Wahl, S.M., Deng, C. and Roberts, A. B. (1999) Nat Cell Biol. 1:260-6).Similarly, the expression of a dominant-negative type II TGF-β receptorin keratinocytes leads to re-epithelialization being again accelerated.Transgenic mice with over expression of TGF-β1 show a better quality ofwound healing with reduced scar formation (Shah, M., Revis, D., Herrick,S., Baillie, R., Thorgeirson, S., Ferguson, M. and Roberts, A. (1999)Am. J. Pathol. 154:1115-24). Similarly, transgenic mice lacking beta3-integrin show elevated levels of TGF-β1 that are associated withenhanced healing and faster fibroblast migration into the wounds(Reynolds, L. E., Conti, F. J., Lucas, M., Grose, R., Robinson, S.,Stone, M., Saunders, G., Dickson, C., Hynes, R. O., Lacy-Hulbert, A. andHodivala-Dilke, K. (2005) Nat. Med. 11:167-74). CD18 knockout miceexhibit reduced TGF-131 expression and have delayed wound healing whichcan be rescued by injecting TGF-β1 into the wound margins (Peters, T.,Sindrilaru, A., Hinz, B., Hinrichs, R., Menke, A., Al-Azzeh, E. A.,Holzwarth, K., Oreshkova, T., Wang, H., Kess, D., Walzog, B., Sulyok,S., Sunderkotter, C., Friedrich, W., Wlaschek, M., Krieg, T. andScharffetter-Kochanek, K. (2005) EMBO J. 24:3400-10).

The elevated levels of TGF-β1 staining that we see on day 2 appear to belargely in elongated fibroblast-like cells at the edge of the wound andin the keratinocytes at the edge of the wound. This is consistent withpreviously-described effects of TGF-β1 on enhanced proliferation andmigration (Postlethwaite, A. E., Keski-Oja, J., Moses, H. L. and Kang,A. H. (1987) J Exp Med 165:251-6), both of which we see in the earlystages of tissue repair. Indeed, the TGF-β1 elevation may be one of thefactors that in these conditions contribute to the promotion of healing,in terms of the increased rate of proliferation and migration offibroblasts and the enhanced rate of collagen synthesis that we see atthese early time points.

Both the active downregulation of Cx43 protein and the action of thewound-associated growth factor TGF-β1 have been reported to activateCollal expression (Cutroneo, K. R. (2003). How is Type I procollagensynthesis regulated at the gene level during tissue fibrosis. J CellBiochem 90, 1-5; Waggett, A. D., Benjamin, M. and Ralphs, J. R. (2006)Eur J Cell Biol. 2006 Jul. 19; [Epub ahead of print]). The enhancedCol1α1 expression and collagen deposition that we see followingtreatment may be related to the increased expression of TGF-β1 at 2days, or to the Cx43 protein downregulation or both.

The wound-associated growth factor, TGF-31, plays a wide variety ofroles at many stages of the wound-healing process. TGF-β1 was at lowlevels on days 1 and 7 with no difference between control and treatedwounds. However, on day 2 after the injury, the expression of TGF-β1 inasODN-treated wounds was significantly increased (P<0.05) compared withcontrol sODN-treated wounds. Immunostaining for TGF-β1 at 2 daysrevealed TGF-β1 positive cells in the dermis both at the wound site andin the adjacent tissues. Most of the cells were round and had theappearance of leucocytes. However, in the Cx43 asODN-treated tissue anadditional TGF-β1 positive cell type could be seen in large numbers atthe dermal margins of the wound. These cells appeared to be elongatedand more fibroblast like in their morphology. Interestingly, in theepidermis of Cx43 asODN treated wounds TGF-β1 appeared to stain muchmore strongly than in control wound epidermis (FIG. 6.) These resultsraise the possibility that increased expression of TGF-β1 mightcontribute to some of the changes we see in wound healing following Cx43asODN treatment.

Whilst TGF-β1 has been suggested to suppress inflammation, it isunlikely to be the main factor leading to the reduced inflammation thatwe observe, as that is already evident on day 1 before the TGF-β1becomes elevated. Similarly, whilst TGF-β1 has been shown to promoteangiogenesis and granulation-tissue maturation at later stages of thewound-healing process we see no elevation of TGF-β1 at these later timepoints; rather it is then at a similar low level to controls. So otherfactors must promote the enhanced maturation of granulation tissue thatwe see following treatment.

These experiments support the implication of TGF-β1 has been implicatedin the in the suppression of inflammation, promotion of angiogenesis andgranulation-tissue maturation of the wound-healing process and indicatesthat modulation of TGF-β1 in combination with other wound treatmentmodalities may be useful in promoting the wound healing process.

Migration and Proliferation

Migration and proliferation of fibroblasts and keratinocytes areindispensable for skin wound healing contributing to both woundcontraction and wound closure. In this study we show enhanced migrationof fibroblasts into the wound site and faster reepithelialisationfollowing Cx43 asODN treatment. These results indicate that Cx43 mayplay an important role in modulating cell movement. Contradictoryresults have been reported on the role of Cx43 in cell movement byothers who reported that in embryonic development, Cx43-deficientproepicardial cells migrated faster than those expressing Cx43 (Li, W.E., Waldo, K., Linask, K. L., Chen, T., Wessels, A., Parmacek, M. S.,Kirby, M. L. and Lo, C. W. (2002). Development 129, 2031-42). However,the same group also previously reported that Cx43 deficient neural crestcells showed decreased rates of migration (Huang, G. Y., Cooper, E. S.,Waldo, K., Kirby, M. L., Gilula, N. B. and Lo, C. W. (1998). J Cell Biol143, 1725-34). This latter finding is consistent with slowed rates ofmigration of retinal neuroepithelial cells that have had communicationperturbed or Cx43 expression diminished (Pearson R., Luneborg, N.,Becker, D. L. and Mobbs P. (2005) J. Neurosci 25 (46), 10803-10814).These differences in the effects of communication on migration mayperhaps reflect the different cell types involved and whether the cellsmigrate independently or in a communicating group.

Faster re-epithelialization and an enhanced rate of granulation tissueformation could also be attributed to the enhanced proliferation in theasODN-treated group, which we find in nascent epidermis and granulationtissue at both 2 days and 7 days after wounding. As with migration ratesthere are mixed reports relating Cx43 expression and proliferation.Cx43-deficient proepicardial cell proliferation is increased, but thisis not seen Cx43-deficient cardiac neural crest cells or in thedeveloping neural retina treated with Cx43 asODNs where proliferation isreduced (Huang, G. Y., Cooper, E. S., Waldo, K., Kirby, M. L., Gilula,N. B. and Lo, C. W. (1998). J Cell Biol 143, 1725-34; Becker, D. L. andMobbs, P. (1999) Exp. Neurology, 156, 326-332; Li, W. E., Waldo, K.,Linask, K. L., Chen, T., Wessels, A., Parmacek, M. S., Kirby, M. L. andLo, C. W. (2002). Development 129, 2031-42). It is interesting that theeffects of Cx43 reduction on proepicardial cells promotes bothproliferation and migration, whereas the effect on neural crest andretinal neuroepithelial cells is to perturb proliferation and migration.Our experiment here show that reducing Cx43 expression with asODNs in afibroblast wound healing assay significantly accelerates their rate ofmigration. The Cx43 protein down regulation may therefore aid the fasterre-epithelialization and fibroblast migration into granulation tissuethat we see here. Alternatively the influence may come from the elevatedlevels of TGF-β1, that we see on day 2 in Cx43 asODN-treated wounds,which has been suggested to enhance cell proliferation and increaserates of fibroblast migration (Mustoe, T. A., Pierce, G. F., Thomason,A., Gramates, P., Sporn, M. B. and Deuel, T. F. (1987). Science 237,1333-6: Postlethwaite, A. E., Keski-Oja, J., Moses, H. L. and Kang, A.H. (1987) J Exp Med 165:251-6) or perhaps a combination of both.

Angiogenesis

Angiogenesis is another central feature of granulation tissue formationand maturation, involving invasion, expansion and then remodeling. Inthis study, blood vessels growing into Cx43 asODN-treated woundgranulation tissue were much more advanced than the controls. On day 5fine blood vessels could be seen entering the granulation tissue of alltreated wounds but no vessels were seen in any control granulationtissue. By day 7 fine blood vessels could be seen throughout thegranulation tissue in the majority of treated wounds but were only seenat the edges of control wounds. Interestingly, the blood vessels incontrol wounds appeared to be thicker at these early stages and so gavea significantly greater area of staining at both 7 and 10 days. By 14days the blood vessels in treated wounds had developed to a greater sizeand appeared to be very similar to controls. Cx43 is known to beinvolved in coronary vasculogenesis and angiogenesis (Walker, D. L.,Vacha, S. J., Kirby, M. L. and Lo, C. W. (2005). Dev Biol 284, 479-98).However, the observation that Cx43 protein levels were similar incontrol and treated wounds by day 7 after injury suggests thatangiogenesis in Cx43 asODN-treated wounds at this stage was most likelyto have been indirectly influenced by the antisense-mediated changesthat we saw at early stages. It is possible that angiogenesis ispromoted by the early elevation in TGF-β1, as this growth factor hasbeen reported to promote angiogenesis (Roberts, A. B., Sporn, M. B.,Assoian, R. K., Smith, J. M., Roche, N. S., Wakefield, L. M., Heine, U.I., Liotta, L. A., Falanga, V., Kehrl, J. H. et al. (1986). Proc NatlAcad Sci USA 83, 4167-71), but the time frames for this do not match.

Granulation Tissue Maturation, Contraction and Cell Death

The area of granulation tissue following Cx43 asODN treatment isconsistently smaller than that of control wounds. This is likely to bedue to several factors. The reduced inflammatory response has severalknock-on effects on subsequent stages of the healing process. Reducedneutrophil invasion will result in reduced damage in surrounding tissue,and most excisional wounds expand in size over the first few days ofhealing as the inflammatory process kicks in whereas treated woundscontract dramatically in the same period. Therefore, reducedinflammation would be expected to result in a significantly smaller areafor the fibroblasts to fill. Because one of the other key effects thatwe have seen is and enhancement of fibroblast proliferation andmigration the smaller wound can be filled much faster and thegranulation tissue can begin to mature significantly faster.

In summary, these studies report our first data led analysis of themechanism underlying the cell biology downstream of Cx43 proteinreduction at wound sites. The local downregulation strongly influencesvery early events in wound healing. In particular, it limits the extentof the inflammatory response and advances the onset and rate ofre-epithelialization and the level and rate of granulation tissueformation. The granulation tissue then has a smaller area to fill anddoes this faster, leading to earlier wound contraction and maturation.This approach clearly offers the potential for new therapies forimproving wound healing in a variety of clinical situations.

Based on these results, experimental downregulation of connexin 43expression at skin wound sites markedly improve the rate and quality ofhealing. The physiological and cell biological aspects of the repairprocess are compared in treatment with and without an exemplaryanti-connexin agent—connexin 43 antisense oligodeoxynucleotide. Treatedwounds exhibited accelerated skin healing with significantly increasedkeratinocyte and fibroblast proliferation and migration. In vitroknockdown of connexin 43 in a fibroblast wound-healing model alsoresulted in significantly faster healing, associated with increased mRNAfor transforming growth factor beta 1, and collagen alpha1 and generalcollagen content at the wound site. Treated wounds showed enhancedgranulation tissue formation and maturation with more rapidangiogenesis, myofibroblast differentiation and wound contraction wasapparently advanced by 2-3 days. Recruitment of both neutrophils andmacrophages was markedly reduced within treated wounds, concomitant withreduced leukocyte infiltration. In turn mRNA levels of chemokine ligand2 and tumor necrosis factor alpha were reduced in the treated wound.These data indicate that under these conditions, reducing connexin 43protein with connexin 43-specific antisense at wound sites early in theskin healing process enhances repair, at least in part, by acceleratingcell migration and proliferation and by attenuating inflammation and theadditional damage it can cause.

Acute downregulation of Cx43 protein at the wound site led to anincrease in keratinocyte proliferation and migration, and in the rate atwhich fibroblasts migrate into the wound and lay down collagen matrix.This correlated with a decrease in neutrophil infiltration and aconcomitant reduction in chemokine ligand 2 (Ccl2) and cytokine tumornecrosis factor alpha (TNF-α) mRNA. Subsequently, a reduced recruitmentof macrophages was seen, perhaps as a consequence of damping down of theinitial inflammatory response. In contrast, increased expression ofTGF-β1 with increased hydroxyproline and collagen type 1α1 was observedin Cx43AsODN treated wounds. Together these modified responses resultedin significantly improved wound healing.

Example 2 Wound Healing in a Human Corneal Limbal Rim Model

Cell denuded pig stroma chimeras were cultured for two weeks and thenassessed using a growth factor/cytokine antibody array (measures proteinlevels of 120 different growth factors/cytokines.

Human limbal rims returned from surgery after excision of the centralcorneas for keratoplasty (transplant) were placed into air liquidinterface organotypic culture and a denuded pig stromal extracellularmatrix inserted into the central region (FIG. 14). The pig stromalmatrix was denuded (cells removed) by freeze-thawing in liquid nitrogen.

The chimeras were grown for up to 2 weeks at which time the human limbalepithelial cells have re-epithelialised the pig stroma with a normal 5-7cell layer fully differentiated epithelium. In addition, human limbalstromal keratocytes have proliferated and migrated across the limbalrim—stromal insert to repopulate the pig stromal matrix with humankeratocytes.

In a further experiment the chimeras were treated with connexin 43specific antisense ODNs at day one. This resulted in a significantincrease in the rate of re-epithelialisation with full epithelialcovering of the pig stroma within 3 days compared with 7 to 14 days forthe controls.

After two weeks in culture the chimeras were cut in half: one half forhistological analysis, and the other for growth factor and cytokinearray analysis. For growth factor and cytokine analysis the centralstromal region (with fully recovered human cell epithelium) andpartially repopulated stroma (human keratocytes) was removed andhomogenised separately from the human limbal rim which contains stemcell populations. The homogenates were run on a growth factor/cytokinearray (FIG. 15) and analysis of growth factor levels performed usingdensitometry analysis of the array membranes. Analysis of key growthfactors revealed a number of interest (FIG. 16 and FIG. 17).

Analysis of growth factor levels in chimeric stroma compared with limbalrims regions of the chimera after two weeks in culture (controlcorneas—not antisense treated) revealed two growth factors of particularinterest (FIG. 16). These were very high levels of IGFBP-2 in thechimeric stroma and higher levels of IGF-1 in the limbal rim. The formeris said to promote cellular migration (needed for the stromalkeratocytes to repopulate the stoma from the limbal rim) and the latteris said to promote cell proliferation (necessary in the limbal rim toprovide the source of cells repopulating the stroma).

Analysis of growth factor levels in chimeric stroma compared with limbalrims regions of the Cx43As ODN treated chimeras after two weeks inculture revealed two growth factors of particular interest (FIG. 17).These were very high levels of IGF-7 in the antisense treated chimericstroma compared with the untreated controls, and higher levels ofIGFBP-2 in both the limbal rim and stroma compared with untreatedcontrols, especially control limbal rims. The former is said to promoteepithelial growth (consistent with the increased re-epithelialisationseen in antisense treated chimeras) and the latter is said to promotecell migration (consistent with the increased epithelial repopulationfrom the limbal rim with antisense treatment).

Example 3

Second degree burns are treated with a spray formulation includinganti-connexin agent, 10 ug/ml of KGF, 30 ng/ml of a PDGF isoform, 10ng/ml IGF-1, and 30 ng/ml of IGFBP-1. The spray is allowed to dry in theair. Re-application is suggested every couple of hours.

Example 4

A suture wound is closed and a topical salve made up of anti-connexinagent, 10% KGF and/or 5% PDGF is applied on the suture before bandaging.Re-application of the salve may be as needed in order to facilitate orimprove wound healing, for example, up to 2-3 times daily for about 4-7days, or as appropriate.

Example 5

A 20% zinc oxide formulation containing an anti-connexin agent and oneor more of 5% KGF, 2.5% PDGF, 10% IGFBP is applied to minor abrasions,sunburns and chafing for faster healing of these wounds.

Example 6

A formulation containing an anti-connexin agent and one or more activeantibiotic ingredients, for example, one or more of bacitracin,neomycin, and polymyxin is prepared. In one such formulation, each gramcontains Polymyxin B Sulfate (5,000 units), Bacitracin Zinc (400 units),and/or Neomycin (3.5 mg). Inactive ingredients of the formulation mayinclude cocoa butter, cottonseed oil, olive oil, sodium pyruvate,tocopheryl acetate, and white petrolatum, in desired amounts.Preferably, the anti-connexin agent is an anti-connexin 43 agent. Suchformulations are applied, for example, to minor cuts, abrasions,sunburns and chafing for faster/improved healing of these wounds.

Example 7

A formulation containing an anti-connexin agent as provided in Example 6is prepared to also contain a topical analgesic (e.g., pramoxine).

Example 8

A formulation containing an anti-connexin agent as provided in Example 6or Example 7 is prepared to contain gramicidin.

Example 9

A formulation containing an anti-connexin agent and one or more activeantifungal ingredients, for example, miconazole nitrate one is prepared.One such formulation contains 2% miconazole nitrate. Inactiveingredients may include propylene glycol 300, polysorbate 20 (and), SDalcohol 40B. The product may formulated as a cream, gel, spray orliquid. Suitable propellants for spray formulations include dimethylether. Preferably, the anti-connexin agent is an anti-connexin 43 agent.Such formulations are applied, for example, to treat athlete's foot,jock itch, and ringworm.

Example 10

This example demonstrates a method for identifying potential cytokinesuseful in the treatment of wounds using a corneal model. The corneacontains dendritic cells known as Langerhans cells, which are activatedin response to injury. Additional inflammatory cells are then recruitedfrom the blood vessels at the limbus. Our model corneas are cultured inisolation from a blood supply, therefore, recruitment of macrophages,T-cells and polymorphonuclear cells is impossible.

The model coreneas were used to determine if the presence of Langerhanscells would be sufficient to illicit an inflammatory response andwhether that response could be distinguished from the healing response.Two matched human corneas were wounded using different techniques. Onecornea was ablated to a depth of 80 μm over a 7 mm diameter using anexcimer laser phototherapeutic keratectomy ablation, a technique whichstimulates very low levels of inflammation. The other cornea was woundedusing a 7 mm circle of filter paper soaked in sodium NaOH for 1 minute,a technique known to induce inflammation. After culturing for 24 hours,the corneas were divided in half (one for immunohistochemistry and onefor cytokine analysis. The cornea was further divided into portionswithin the healing zone and outside the healing zone before processingfor cytokine array analysis.

An inflammatory response was identified in both corneas but it wasenhanced within the alkali burn. Cytokine profiles between the twogroups due to the greater inflammation within the alkali burn sample andbecause healing commenced faster in the lasered cornea as less removalof necrotic tissue is required. FIG. 18 shows a group of cytokinesincreased in both wounds but far more so in the inflammatory woundmodel. This group included interleukins. FIG. 19 shows a set ofcytokines which were increased in both models but more so in the lowinflammation model and thus represent cytokines to target for improvedhealing.

Efficacy of various cytokines in the wound healing model is determinedby assessing the effect of the cytokines augmentation in a wound healingmodel. In addition, the abilities of various cytokines of interest toincrease the rate of wound healing and to improve quality of healing areassessed.

Example 11

This example demonstrates a method for dertermining cytokines fortherapeutic use. Cytokines may be selected for use herein based on theiraltered levels as induced by Cx43AsODN treatment. These cytokines canhave an effect on corneal wound healing, for example, when they areadministered exogenously in a keratoplasty model of wound healing.

Model: Paired human limbal rims with inserted pig stromal implants aretested in cultured keratoplasty model for alterations in the cytokinelevels. The tissue level of the added cytokine is gauged by array data.For temporal study, cytokines are added at the onset of the healingprocess. Optimal treatment periods are determined with additional datapoints if needed. In the case wherein the healing follows the same timecourse as the Cx43AsODNs model, then the culture times are assessed for3 and 7 days. Healing time course is determined using dark fieldmicroscopy and adjusted as necessary.

Technique: 5 pairs of limbal rims per cytokine are used in escalatingdoses to establish a dose response curve.

The same methods for investigation of corneal wound healing are employedas stated above in Example 9, except that the epithelial plug that formsin unsutured corneal wound healing are used to isolate epithelial woundhealing from stromal wound healing. Factors that influence epithelialrecovery are assessed independently of the influence of stromal cells.In some instances it might be preferable to increase corneal epithelialwound healing to preserve the barrier protection, but not to increasestromal wound healing which might induce haze.

Cytokines of interest in corneal wound healing are identified.Candidates include, for example, IGF and IGFBP2 from normal tissuestudies and FGF-7 from antisense studies.

All patents, publications, scientific articles, web sites, and otherdocuments and materials referenced or mentioned herein are indicative ofthe levels of skill of those skilled in the art to which the inventionpertains, and each such referenced document and material is herebyincorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such patents, publications, scientific articles,web sites, electronically available information, and other referencedmaterials or documents.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,any of the terms “comprising”, “consisting essentially of”, and“consisting of” may be replaced with either of the other two terms inthe specification. Also, the terms “comprising”, “including”,containing”, etc. are to be read expansively and without limitation. Themethods and processes illustratively described herein suitably may bepracticed in differing orders of steps, and that they are notnecessarily restricted to the orders of steps indicated herein or in theclaims. It is also that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Under no circumstances may thepatent be interpreted to be limited to the specific examples orembodiments or methods specifically disclosed herein. Under nocircumstances may the patent be interpreted to be limited by anystatement made by any Examiner or any other official or employee of thePatent and Trademark Office unless such statement is specifically andwithout qualification or reservation expressly adopted in a responsivewriting by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1-66. (canceled)
 67. A method for promoting or improving healing of optic nerve or eye tissue in a subject, comprising administering to the subject an anti-connexin agent simultaneously with, separately from or sequentially with a medicament comprising a therapeutically effective amount of an antagonist of VEGF, wherein the connexin agent is an anti-connexin 43 polynucleotide or an anti-connexin 43 peptide or peptidomimetic.
 68. A pharmaceutical composition for use in promoting or improving healing of optic nerve or eye tissue in a subject, comprising therapeutically effective amounts of an anti-connexin 43 agent and an antagonist of VEGF, and wherein the connexin agent is an anti-connexin 43 polynucleotide or an anti-connexin 43 peptide or peptidomimetic.
 69. The composition according to claim 68, wherein said anti-connexin 43 polynucleotide molecule is a modified or unmodified antisense polynucleotide molecule.
 70. The composition according to claim 68, wherein said antisense polynucleotide molecule comprises any one of a sequence selected from SEQ ID NOS:1 to
 3. 71. The composition according to any one of claims 68-70, wherein the composition comprises about 0.1 to about 1000 micrograms of said anti-connexin 43 antisense polynucleotide molecule.
 72. The composition according to any one of claims 68-70, wherein the composition comprises about 0.001 to about 100 milligrams of said anti-connexin 43 peptide or anti-connexin 43 peptidomimetic.
 73. The pharmaceutical composition according to any one of claims 68-70, wherein the pharmaceutical composition is formulated for sustained release.
 74. The pharmaceutical composition according to claim 71, wherein the pharmaceutical composition is formulated for sustained release.
 75. The pharmaceutical composition according to claim 72, wherein the pharmaceutical composition is formulated for sustained release.
 76. A pharmaceutical composition for use in promoting or improving healing of optic nerve or eye tissue in a subject, formulated as a gel, comprising: (i) 0.1 to 1000 micrograms of an anti-connexin 43 peptide or peptidomimetic; and (ii) an antagonist of VEGF.
 77. The pharmaceutical composition of claim 76, wherein said composition is formulated for instillation.
 78. The pharmaceutical composition according to any one of claim 76 or 77, wherein said composition is formulated for sustained release.
 79. The pharmaceutical composition according to any one of claim 77 or 77, wherein said composition comprises 0.1 to 100 milligrams of said an antagonist of VEGF.
 80. An article of manufacture for use in promoting or improving healing of optic nerve or eye tissue in a subject, comprising package material containing a pharmaceutical composition as defined in any one of claims 68-70, together with instructions for use.
 81. An article of manufacture for use in promoting or improving healing of optic nerve or eye tissue in a subject, comprising package material containing a pharmaceutical composition as defined in claim 71, together with instructions for use.
 82. An article of manufacture for use in promoting or improving healing of optic nerve or eye tissue in a subject, comprising package material containing a pharmaceutical composition as defined in claim 72, together with instructions for use.
 83. An article of manufacture for use in promoting or improving healing of optic nerve or eye tissue in a subject, comprising package material containing a pharmaceutical composition as defined in claim 73, together with instructions for use.
 84. An article of manufacture for use in promoting or improving healing of optic nerve or eye tissue in a subject, comprising package material containing a pharmaceutical composition as defined in any one of claim 76 or 77, together with instructions for use.
 85. An article of manufacture for use in promoting or improving healing of optic nerve or eye tissue in a subject, comprising package material containing a pharmaceutical composition as defined in claim 78, together with instructions for use.
 86. An article of manufacture for use in promoting or improving healing of optic nerve or eye tissue in a subject, comprising package material containing a pharmaceutical composition as defined in claim 79, together with instructions for use.
 87. An article of manufacture according to claim 76, wherein the anti-connexin peptide or anti-connexin peptidomimetic comprises SEQ.ID.NO:19 or SEQ.ID.NO:35
 88. The pharmaceutical composition according to claim 72, comprising about 1 to about 100 micrograms of the peptide.
 89. A method according to claim 67, wherein said antisense polynucleotide has from about 15 to about 35 nucleotides and is sufficiently complementary to connexin 43 mRNA to form a duplex having a melting point greater than 20° C. under physiological conditions.
 90. A method according to claim 67, wherein the connexin 43 polynucleotide has from about 15 to about 35 nucleotides and has at least about 70 percent homology to an antisense sequence of connexin 43 mRNA.
 91. A method according to claim 67, wherein the anti-connexin agent and the VEGF antagonist are administered simultaneously.
 92. A method according to claim 67, wherein the anti-connexin agent and the VEGF antagonist are not administered simultaneously.
 93. A method according to claim 67, wherein the anti-connexin agent and the VEGF antagonist are administered sequentially.
 94. A method according to claim 67, wherein the anti-connexin agent is administered first.
 95. A method according to claim 67, wherein the VEGF antagonist is administered first.
 96. The pharmaceutical composition according to any one of claims 68-70, wherein the pharmaceutical composition is formulated in a matrix. 